Receptor specificity of the influenza virus hemagglutinin modulates sensitivity to soluble collectins of the innate immune system and virulence in mice

Virology xxx (2011) xxx–xxx

YVIRO-06105; No. of pages: 11; 4C: 5

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Receptor specificity of the influenza virus hemagglutinin modulates sensitivity tosoluble collectins of the innate immune system and virulence in mice

Michelle D. Tate a, Andrew G. Brooks a, Patrick C. Reading a,b,⁎a Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria 3010, Australiab WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, Australia

⁎ Corresponding author at: Department of Microbiologof Melbourne, Melbourne, Victoria 3010, Australia. Fax:

E-mail addresses: [email protected] ([email protected] (A.G. Brooks), preading@unim

0042-6822/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.virol.2011.01.035

Please cite this article as: Tate, M.D., et acollectins of the innate immune system ...,

a b s t r a c t

Article history:Received 19 November 2010Returned to author for revision5 January 2011Accepted 28 January 2011Available online xxxx

Keywords:InfluenzaMouseHemagglutininSialic acidVirulence

The hemagglutinin (HA) glycoprotein of influenza virus binds to cell surface sialic acid (SA) to initiateinfection. In this study, a mutant of influenza A virus strain BJx109 (H3N2) was plaque-purified from the lungsof virus-infected mice that had been depleted of airway macrophages. Sequence analysis identified a singleamino acid substitution (S186I) in the vicinity of the receptor-binding site of HA. This substitution wasassociated with enhanced binding to α(2,3)-Gal-linked SA and an increased ability to infect murine airwayepithelial cells. Mutant viruses were less sensitive to neutralization bymouse airway fluids and less efficient intheir ability to infect murine macrophages. Moreover, infection of mice with viruses bearing the S186Isubstitution led to severe disease, characterized by enhanced virus replication, lung pathology and pulmonaryedema. Together, these studies confirm that residue 186 of H3 subtype viruses is a critical determinant ofvirulence in a mouse model of influenza infection.

y and Immunology, University+61 39347 1540.D. Tate),elb.edu.au (P.C. Reading).

l rights reserved.

l., Receptor specificity of the influenza viruVirology (2011), doi:10.1016/j.virol.2011.01.

© 2011 Elsevier Inc. All rights reserved.

Introduction

The influenza hemagglutinin (HA) glycoprotein plays a critical rolein initiating cellular infection by recognizing sialic acid (SA) expressedon cell surface glycoproteins and glycolipids (reviewed by Skehel andWiley, 2000). The globular head of HA contains the receptor-bindingsite (RBS), a shallow pocket of highly conserved amino acids thatinteract with sialylated receptors. In nature, SA is generally attachedto the penultimate galactose residues of oligosaccharide side chainsby α(2,3)-Gal or α(2,6)-Gal linkages (Skehel and Wiley, 2000). TheHA of human influenza viruses shows a preference for binding toα(2,6)-Gal-linked SA (Connor et al., 1994; Rogers and Paulson, 1983),which is abundantly expressed on non-ciliated cells in the humanrespiratory tract (Matrosovich et al., 2004). In contrast, avianinfluenza viruses prefer α(2,3)-Gal-linked SA which is expressed onepithelial cells in the gastro-intestinal tract of numerous avian species,including chickens, turkeys andmigratory ducks (Ito et al., 1998; Pillaiand Lee, 2010).

Amino acids in and around the RBS of HA determine the specific SAlinkages that will be recognized by a particular virus strain. Althoughthe RBS is structurally conserved among a number of HA subtypes,studies have identified specific residues directly within, as well as inthe vicinity of the RBS, that are critical for SA binding and receptor

specificity (reviewed by Russell et al., 2006; Skehel and Wiley, 2000).In particular, amino acids 145 (Matrosovich et al., 1998; Ryan-Poirierand Kawaoka, 1991), 186 (Gambaryan et al., 1999; Matrosovich et al.,1998; Matrosovich et al., 1997; Widjaja et al., 2006), 190 (Gambaryanet al., 1999; Nobusawa et al., 2000), 193 (Medeiros et al., 2001;Medeiros et al., 2004), 225 (Matrosovich et al., 1997), 226 (Pekoszet al., 2009; Vines et al., 1998) and 228 (Matrosovich et al., 1997;Pekosz et al., 2009; Vines et al., 1998) are important in modulating thereceptor specificity of H3 subtype viruses.

Mice are not naturally infected with influenza virus and intranasalinoculation with human influenza viruses generally does not result insevere disease (Hawgood et al., 2004; Reading et al., 1997; Sweet andSmith, 1980; Vigerust et al., 2007), although H5N1 viruses and the 1918pandemic H1N1 virus induce disease in mice without prior adapta-tion (Gao et al., 1999; Perrone et al., 2008; Tumpey et al., 2005). Se-quential passage of human isolates in mouse lung results in selection ofvirus mutants with increased replication efficiency that are capableof inducing viral pneumonia similar to that observed in humans (Sweetand Smith, 1980). In the lower respiratory tract of mice, the expres-sion of α(2,3)-Gal-linked SA predominates over α(2,6)-Gal-linked SA(Glaser et al., 2007; Ning et al., 2009). Adaptation of human influenzaviruses to growth in mouse lung can be associated with a switch inreceptor specificity to enhance binding to α(2,3)-Gal-linked SA(Hensley et al., 2009; Keleta et al., 2008; Smeenk et al., 1996).

Surfactant protein (SP)-D is an innate immune protein of thecollectin superfamily present in airway secretions that binds tomannose-rich glycans on influenza virus HA and/or neuraminidase(NA) to mediate a range of antiviral activities (reviewed by Crouch

s hemagglutinin modulates sensitivity to soluble035

Table 1Receptor specificity of BJx109 mutant viruses.

Virus Hemagglutination titera

Native Asialo α(2,3)-SA α(2,6)-SA

BJWT 64 b1 b1 32BJpp1 64 b1 32 32BJpp2 64 b1 32 32BJpp3 64 b1 32 32PR8 64 b1 32 b1

a BJWT, BJpp1-BJpp3 or PR8 viruses were compared for their ability to agglutinatedesialylated and enzymatically resialylated erythrocytes. Human erythrocytes weredesialylated with bacterial sialidase from C. perfringens for 1 h at 37 °C andenzymatically resialylated with Neu5Ac in the presence of either α(2,3)-sialyltransfer-ase or α(2,6)-sialyltransferase as described in Materials and methods. Native humanerythrocytes were included for comparison. HA assays were performed using standardprocedures and data are expressed as HAU. PR8 was included due to its knownspecificity for α(2,3)-Gal-linked SA.

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andWright, 2001). Previous studies have noted a correlation betweenthe sensitivity of different influenza viruses to neutralization by SP-Dand their ability to replicate in mouse lung. Highly glycosylated virusstrains that were neutralized by SP-D did not replicate efficiently inthe lungs of immunocompetent mice (Reading et al., 1997; Vigerustet al., 2007), but replicated to high titers in SP-D-knockout mice(Vigerust et al., 2007). β-Inhibitors in mammalian serum and lungfluids are mannose-binding lectins of the collectin superfamily(Anders et al., 1990) and adaptation of human H1 viruses to growthin mouse lung was associated with development of resistance to β-inhibitors (Briody et al., 1955; Chu, 1951; Shilov and Sinitsyn, 1994).Furthermore, β-resistant H3 subtype viruses selected in the presenceof bovine serum (a rich source of the bovine collectin conglutinin)showed enhanced virulence in mice (Hartley et al., 1997). Additionalfactors, including changes in HA/NA balance (Wagner et al., 2002) andpoint mutations in genes encoding internal components of the virion,including PB1-F2 (Conenello et al., 2007), M1 (Brown et al., 2001;McCullers et al., 2005) and polymerase genes (Gabriel et al., 2005;Hatta et al., 2001), also contribute to virulence in mice.

We have recently demonstrated that depletion of airway macro-phages (Mϕ) during infection of C57BL/6 mice with the virus strainBJx109 (H3N2) was associated with enhanced disease severity,characterized by increased virus replication and pulmonary inflam-mation (Tate et al., 2010b). Plaques formed on MDCK cell monolayersinoculated with lung homogenates from BJx109-infected mice de-pleted of Mϕ (via intranasal treatments with clodronate-loadedliposomes (CL-LIP)) were plaque-purified, amplified once in eggs andused to inoculate untreated C57BL/6 mice. Each of the plaque-purifiedviruses showed enhanced virulence in mice. The studies describedherein have investigated the mechanism/s underlying the enhancedvirulence of BJx109 viruses in mice.

Results

Isolation of virus mutants of BJx109 from the lungs of mice depleted ofMϕ

In a previous study, we examined the role of Mϕ following in-fection of mice with influenza virus BJx109 (Tate et al., 2010b).Treatment of virus-infected mice with saline-loaded liposomes(SL-LIP) was associated with mild disease, whereas treatment ofmice with CL-LIP (to deplete airway Mϕ) led to progressive weightloss and death. At day 7 post-infection, lungs from BJx109-infectedmice treated with CL-LIP were plaqued on MDCK cell monolayers. Noinfectious virus was recovered from the lungs of SL-LIP-treated miceat this time, whereas high levels of virus were recovered from micetreated with CL-LIP (log10PFU 4.63±0.03) and 5 plaques (from 3different mice) were picked, amplified once in eggs and analyzed.Allantoic fluid containing either wild-type BJx109 (BJWT; as used forintranasal inoculation of mice) or plaque-purified BJx109 (BJpp1-BJpp5)were inoculated onto MDCK cell monolayers and plaque size wasdetermined 3 days later. BJpp1-BJpp5 formed plaques that were smaller(~200 μm diameter from 10 individual plaques per sample) than BJWT

(~1000 μm). These differences in plaque size were likely to reflectdifferences in the properties of the HA and/or NA glycoproteins of thevirus as these play a major role in facilitating the cell to cell spread ofvirus (Baigent and McCauley, 2001; Suzuki et al., 2005).

The small plaque phenotype of BJx109 mutant viruses is associated witha single amino acid change in HA

RNA was extracted from allantoic fluid containing BJWT and BJpp1-BJpp5 and the HA and NA genes were amplified by PCR, sequenced andaligned. The deduced amino acid sequence of the NA from BJWT andBJpp1-BJpp5 was identical. In contrast, each small plaquemutant differedfromBJWTby a singlebase substitution (A814→T) inHA1,which resulted

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

in substitution of Ser with Ile at residue 186 (S186I). Residue 186 islocated on the head of HA in the vicinity of the RBS but does not directlyinteract with SA. However, it does interact with residues 190 and 228,which form hydrogen bonds with the C9 hydroxyl group of thepolyhydroxyl tail of Neu5Ac and thus mutations at this position havepotential to indirectly alter the conformation of the receptor-bindingsite (Eisen et al., 1997; Sauter et al., 1992; Weis et al., 1988). BJx109viruses carrying the S186I substitution will be referred to hereafter asBJ186.

BJ186 mutants show an enhanced ability to recognize α(2,3)-Gal-linkedSA

Amino acid changes in the RBS of HA are associated with dif-ferences in ability to bind α(2,3)-Gal and α(2,6)-Gal-linked SA(reviewed by Russell et al., 2006; Skehel and Wiley, 2000). Therefore,to assess receptor specificity we first compared BJWT and BJ186 for theirability to agglutinate erythrocytes from different species. No majordifferences were observed in the ability of viruses to agglutinatehuman, chicken, turkey or guinea pig erythrocytes (data not shown),which express differing levels of α(2,3)-Gal-linked and α(2,6)-Gal-linked SA (Ito et al., 1997a; Medeiros et al., 2001). Horse erythrocytesexpress only α(2,3)-Gal-linked Neu5Gc (Eylar et al., 1962; Ito et al.,1997a; Suzuki et al., 1985) and were agglutinated by BJ186 (HAU=4–8 HAU), but not by BJWT (HAU=b1).

Next, human erythrocyteswere desialylated using bacterial sialidaseand enzymatically resialylated to express either α(2,3)-Gal- or α(2,6)-Gal-linked Neu5Ac. BJWT agglutinated erythrocytes expressing α(2,6)-Gal-, but not α(2,3)-Gal-linked Neu5Ac (Table 1). In contrast, BJ186viruses agglutinated erythrocytes bearing either α(2,3)-Gal or α(2,6)-Gal-linkedNeu5Ac, suggesting that S186I results in enhanced binding toα(2,3)-Gal-linkedNeu5Ac,while retainingbinding toα(2,6)-Gal-linkedNeu5Ac. An additional property of the viral HA that could influencethe efficiency of viral replication and therefore virulence is the pH atwhich fusion of the viral envelope with the endosomal membraneoccurs. Using themethod of Doms et al. (1986), we did not observe anysignificant differences in the optimal pH atwhich BJWT and BJ186 virusesmediated lysis of human erythrocytes (data not shown).

Enhanced ability of BJ186 to bind and infect murine airway epithelial cells

To investigate whether changes in receptor specificity of BJ186viruses impacted on ability to infect epithelial cells, monolayers ofLA-4 cells (a mouse airway epithelial cell line (Stoner et al., 1975))were infected with increasing doses of BJWT or BJ186 and, after 1 h,monolayers were washed, incubated for 7 h and fixed and stained byimmunofluorescence for expression of viral NP (Fig. 1A). At a highinoculum dose (106 PFU), BJWT and BJ186 viruses infected LA-4 cells

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Fig. 1. Enhanced ability of BJ186 to bind and infect mouse LA-4 epithelial cells.(A) Monolayers of LA-4 cells were incubated with increasing doses of BJWT or BJpp1-BJpp3 viruses. After 1 h, monolayers were washed, incubated a further 7 h and fixed andstained by immunofluorescence for expression of newly synthesized viral NP.(B) Monolayers of LA-4 cells were incubated with 106 PFU of BJWT or BJpp1-BJpp3viruses for 5 min, washed, and incubated a further 7 h. At this time, cells were fixed andstained by immunofluorescence for expression of viral NP. Data in A/B represent themean percent infection (±1 SD) from aminimum of 4 independent fields per chamber.*=BJWT is significantly reduced compared to all other groups, pb0.01, one-wayANOVA. (C) Ability of viruses to bind to and elute from the epithelial cell surface.Duplicate tubes of formaldehyde-fixed LA-4 epithelial cells were mixed with 128 HAUof each virus for 1 h on ice. After virus adsorption, one sample was held at 4 °C (‘4°C’),while the duplicate tube was transferred to 37 °C for 30 min (‘37°C’). A third tube withno cells received virus alone (‘Control’) and was held on ice throughout. Followingcentrifugation, virus in each of the supernatants was quantified by hemagglutinationassay. Data shown are the mean±1 SD HAU from 3 independent experiments.(D) Monolayers of LA-4 cells were infected with 0.01 PFU/cell of BJWT or BJpp1 virus andat time-points indicated, cell supernatants were removed and clarified, and titers ofinfectious virus in cell supernatants were determined by plaque assay on MDCK cells.Data represent the mean virus titer±1 SD from triplicate samples and arerepresentative of 2 independent experiments. *=significantly different to BJWT virus,pb0.05, Student's t test.

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Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

to equivalent levels; however, as the inoculum was reduced BJ186viruses infected cells more efficiently. These findings are consistentwith reports that α(2,3)-Gal-linked SA is the predominant linkageexpressed on murine airway epithelial cells (Glaser et al., 2007; Ninget al., 2009) and with our findings that the α(2,3)-Gal-specific plantlectin Maackia amurensis agglutinin (MAA) binds more avidly to LA-4cells than α(2,6)-Gal-specific Sambucus nigra agglutinin (SNA) (Tateet al., 2011).

Next, we investigated if reducing the time of exposure to virusinoculum might also reveal differences in the ability of BJWT and BJ186to infect epithelial cells. LA-4 cells were incubated for 5 min with 106

PFU of BJWT or BJ186, washed and the percent of virus-infected cellswas determined 8 h later. Incubation of cells with BJWT inoculum for60 min resulted in ~90% infection of LA-4 cells (Fig. 1A), whereasinoculation with an equivalent dose for 5 min resulted in ~40%infection (Fig. 1B). In contrast, inoculation of LA-4 cells with 106 PFUof BJ186 viruses for either 60 min (Fig. 1A) or 5 min (Fig. 1B) resultedin equivalent (~90%) levels of infection. Thus, S186I is associated withenhanced ability to infect murine airway epithelial cells.

As the balance of HA and NA activity can affect the efficiency ofvirus entry (Wagner et al., 2002), we next compared the ability ofviruses to bind and elute from the surface of LA-4 epithelial cells. Allviruses bound efficiently to paraformaldehyde-fixed LA-4 cells at 4 °C(as indicated by a reduced HA titer) but differed in their ability toelute from the cell surface when shifted to 37 °C (Fig. 1C). BJWT elutedefficiently from LA-4 cells at 37 °C; however, BJ186 viruses were notreleased. Thus, BJ186 viruses bind more avidly to LA-4 cells such thatthe activity of the viral NA is less effective at releasing virus from thecell surface.

We usedmultistep growth curves to compare the ability of BJWT andBJ186 to replicate in LA-4 cells (Fig. 1D). Although BJ186 viruses weremore efficient in their ability to infect LA-4 cells, levels of infectious virusin cell supernatants at 16, 24 and 48 h were significantly reducedcompared to BJWT in 2 independent experiments.

Reduced ability of BJ186 to infect murine airway Mϕ

Influenza A virus infection of airway epithelial cells results in pro-ductive virus replication. In contrast, infection of mouse Mϕ is gen-erally a component of innate defense as virus replication is abortiveand Mϕ respond by secreting pro-inflammatory mediators (Hofmannet al., 1997; Rodgers and Mims, 1981; Tate et al., 2010b; Wells et al.,1978), although some humanMϕ populations can support productiveinfection (Cheung et al., 2002; Perrone et al., 2008). Murine Mϕexpress predominantly α(2,6)-Gal-linked SA and the preference ofBJx109HA forα(2,6)-Gal-linked SA is an important factor contributingto its ability to infect Mϕ efficiently (Tate et al., 2011). We hy-pothesized that the changes in receptor preference of BJ186 virusesmight also affect their ability to infect murine Mϕ. As airway Mϕ andperitoneal Mϕ show a similar susceptibility to infection by differentinfluenza A viruses (Reading et al., 2000; Reading et al., 2010; Tateet al., 2010b), we compared the ability of BJWT and BJ186 viruses toinfect adherence-purified peritoneal Mϕ. At all inoculum doses tested,BJ186 viruses were less efficient in their ability to infect Mϕ (Fig. 2A).Furthermore, when the exposure to inoculum was reduced from60 min (Fig. 2A) to 5 min (Fig. 2B), BJWT was again more efficient thanBJ186 in mediating infection of Mϕ.

Neutralization of BJWT and BJ186 viruses by mouse BAL

The HA of BJx109 is highly glycosylated and this virus is very sen-sitive to SP-D-mediated neutralization (Reading et al., 1997). Previousstudies have demonstrated that loss of glycan/s from HA leads toincreased resistance to SP-D and enhanced virulence in mice (Hartleyet al., 1997; Hawgood et al., 2004; Reading et al., 2009; Vigerust et al.,2007). As SP-D represents the major neutralizing activity in mouse

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Fig. 2. BJ186 viruses show reduced ability to infect murineMϕ. (A) Monolayers of PEC Mϕwere incubatedwith increasing doses of BJWT or BJpp1- BJpp3 viruses. After 1 h,monolayerswerewashed, incubated a further 7 h and then fixed and stained by immunofluorescencefor expression of viral NP. Data represent the mean percent infection (±1 SD) from aminimumof 4 independentfields per chamber. (B)Monolayers of PECMϕwere incubatedwith 106 PFU of BJ WT or BJpp1-BJpp3 viruses for 5 min, washed, and incubated for a further7 h. At this time, cells were fixed and stained by immunofluorescence for viral NP. Datarepresents the mean percent infection (±1 SD) from a minimum of 4 independent fieldsper chamber. For panels A and B, *=BJWT is significantly increased compared to all othergroups, pb0.01, one-way ANOVA.

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airwayfluids (Readinget al., 1997; Tate et al., inpress),wecompared thesensitivity of BJx109viruses toneutralizationbymousebronchoalveolarlavage (BAL) fluid. First, dilutions of BAL were incubated with virus and

Fig. 3. Neutralization of BJWT and BJ186 by mouse airway fluids. (A) BJWT or BJpp1-BJpp3 viruincubated for 30 min at 37 °C and added to LA-4 cell monolayers. The amount of infectious virof neutralization by mannan (+mannan) was examined by adding mannan (to a final conceof BAL in TBS containing 10 mM CaCl2 (Buffer) were added to LA-4 cell monolayers and incincubation for 1 h at 37 °C, the inoculum was removed and the amount of infectious virus rneutralization by mannan (+mannan) was examined by adding mannan (to a final concentrindicates dilutions at which BJWT is significantly more sensitive to BAL compared to BJpp vi

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

the amount of infectious virus remaining was determined on LA-4 cellmonolayers (Fig. 3A, upper panel). BAL fluids neutralized BJWT and BJ186to equivalent levels and a significant proportion of the neutralizingactivity was reversed by the inclusion of mannan (Fig. 3A, lower panel).These findings are consistent with SP-D (or a related mannose-bindinglectin) being the major neutralizing activity against BJx109 in mouseBAL. The S186I substitution did not result in loss or gain of N-linkedglycosylation onHA, consistentwith ourfindings that BJWT and BJ186 areequally sensitive to neutralization.

We hypothesized that in the lung, the enhanced binding of BJ186viruses to mouse airway epithelial cells might increase resistance toneutralization by innate immuneproteins inmouse lung.We thereforeperformed a modified neutralization assay in which dilutions of BALwere added to LA-4 cell monolayers prior to the addition of virus, in aneffort to mimic the microenvironment of the lung where the res-piratory epithelium would be lined by pulmonary surfactant. Com-pared to BJWT, we consistently observed a reduced sensitivity of BJ186to neutralization bymouse BAL (Fig. 3B, upper panel).Mannanblockedthe neutralizing activity of mouse BAL against BJWT in the modifiedneutralization assay (Fig. 3B, lower panel).

Enhanced virulence of BJ186 viruses in mice is associated with increasedvirus replication

Mice infectedwith 105 PFU of plaque-purified viruseswereweigheddaily and monitored for signs of disease. Infection with BJWT did notinduce significant weight loss over the 10-day monitoring period,whereas BJ186-infected mice lost weight rapidly and all mice wereeuthanized 7–8 days post-infection (Fig. 4A). At the time of death, BJ186-infected mice displayed hunched posture and laboured breathing,indicative of viral pneumonia. A number of parameters associated withinflammation anddiseasewere analyzed inB6mice infectedwith eitherBJWT or BJ186. As all pp BJ186 viruses showed enhanced virulence(Fig. 4A),wehaveused a single BJ186 virus (BJpp1, hereafter referred to asBJ186) to study these parameters in detail.

Mice infected with 105 PFU of BJWT or BJ186 were killed at days 3and 7 post-infection and titers of infectious virus were determined inthe upper (nasal tissues) and lower (lung) respiratory tract (Fig. 4B).At day 3, viral titers were significantly higher in both the lungs (upper

ses were mixed with dilutions of mouse BAL in TBS containing 10 mM CaCl2 (Buffer),us remaining was determined by fluorescent focus assay at 8 h post-infection. Inhibitionntration of 10 mg/mL) to BAL dilutions 20 min before the addition of virus. (B) Dilutionsubated for 20 min at 37 °C before the addition of BJWT or BJpp1-BJpp3 viruses. Followingemaining was determined by fluorescent focus assay at 8 h post-infection. Inhibition ofation of 10 mg/mL) to BAL dilutions 20 min before the addition of virus. The dashed lineruses (pb0.01, one-way ANOVA).

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Fig. 4. Intranasal infection of mice with BJ186 viruses is associated with increasedvirulence and virus replication. (A) Groups of 5 mice were infected with 105 PFU of BJWT

or BJpp1-BJpp3. Mice were observed daily for weight loss (upper panel). Mice which hadlost ≥25% of their original body weight were euthanized. Data represents the mean %weight change±1 SEM. Survival plots are shown (lower panel). (B) Groups of 5 micewere infected with 105 PFU of BJWT or BJpp1 (BJ186) and at days 3 and 7 post-infectionmice were killed and titers of infectious virus in lung (upper panel) and nasal tissuehomogenates (lower panel) were determined by plaque assay on MDCK cells. Barsrepresent the mean viral titer from a group of 5 mice±1 SD. The detection limit of theassay (0.9) is indicated by the dotted line. *=virus titers from BJ186-infected mice weresignificantly higher than those from BJWT-infected mice, pb0.01, one-way ANOVA.

Fig. 5. Infection of mice with BJ186 virus is associated with severe pulmonary pathology.Groups of 10 B6mice were infected with 105 PFU of BJWT or BJ186 (BJpp1) and were killedand analyzed at day 7 post-infection. (A) Representative images of inflammation inH&E-stained lung sections. Lung sections from naïve mice and mice 5 days afterintranasal infection with 105 PFU of PR8 are included for comparison. Images are shownat 10× magnification. (B) Histopathological scores for lung sections from BJx109-infected mice. Lung sections were scored blind for alveolitis and peribronchiolarinflammation from 0 to 5. Data shown represent scores from individual mice (asindicated by circles) and median values (as indicated by bar) obtained from 1 of 3independent readers. Samples were compared for statistical significance using theKruskal–Wallis test (nonparametric). For each reader, significant differences wereobserved in histopathology scores between mice infected with BJWT compared to BJ186mice (pb0.05, for alveolitis and peribronchiolar inflammation).

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panels) and nasal tissues (lower panels) of mice infected with BJ186(~500-fold and ~30-fold increases in the lungs and nasal tissues,respectively). By day 7, BJWT-infected mice had cleared virus from theairways, whereas high titers were recovered from the lungs and nasaltissues of BJ186-infected mice.

Infection of B6 mice with BJ186 is associated with the development ofsevere pulmonary inflammation

We examined lung sections from mice infected 7 days previouslywith either BJWT or BJ186. Lung sections from naïve animals or mice

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

infected 5 days earlier with the mouse-adapted PR8 virus wereincluded for comparison. Few inflammatory cells were evident insections from naïve (Fig. 5A-i) or BJWT-infected mice (Fig. 5A-ii),whereas infectionwith BJ186 (Fig. 5A-iii) was associatedwith an influxof inflammatory cells and a pulmonary pathology similar that asso-ciated with severe disease during PR8 infection (Tate et al., 2010b)(Fig. 5A-iv). Lung sections were blinded, randomized and scored by 3independent readers for alveolitis (Fig. 5B-i) and peribronchiolarinflammation (Fig. 5B-ii). Levels of alveolitis and peribronchiolarinflammation scored were not statistically different (i) betweennaïve and BJWT-infected mice, or (ii) between BJ186 and PR8-infectedmice; however, both alveolitis and peribronchiolar inflammationwere significantly higher in mice infected with BJ186 compared tomice infected with BJWT.

Increased cellularity and production of inflammatory mediators in theairways of mice infected with BJ186

Flow cytometry was used to analyze the cellular infiltrate in theairways of mice infected with either BJWT or BJ186. Infection with BJWT

induced a modest recruitment of leukocytes to the airways at days 3and 7 post-infection (Fig. 6A), whereas BAL cell numbers were sig-nificantly higher from mice infected with BJ186. Neutrophils pre-dominated in the airways 3 days after infection with BJ186 andnumbers of NK cells, total T cells, CD8+ T cells and B220+ lymphocytes

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Fig. 6. Increased cellularity and production of inflammatory mediators in the airways ofBJ186-infectedmice. Groups of 5micewere infectedwith 105 PFUof BJWT or BJ186. At days 3and 7 post-infection mice were killed and analyzed. Naïve mice were included forcomparison. (A) Total numbers of BAL cells were determined by viable cell counts.*=significantly increased compared to BJWT-infected mice, pb0.01, one-way ANOVA.(B) BAL cells were examined by flow cytometry for the presence of neutrophils (Gr-1high),NK cells (NK1.1+ TCRβ−), B cells (B220+), T cells (TCRβ+) and CD8+ T cells (CD8+

TCRβ+). Bars represent themean cell number±1 SD. *=cell numbers fromBJ186-infectedmice that significantly higher compared to BJWT-infected mice, pb0.05, one-way ANOVA.(C) Concentrations of IL-6, IFN-γ, MCP-1 and TNF-α in cell-free BAL supernatants weredetermined by cytokine bead array at day 7 post-infection. Bars represent the meanconcentration (pg/ml) and individualmice are shownas circles. The detection limit of eachmediator is 5 pg/ml and is indicated as a dotted line. *=levels from BJ186-infected micethat were significantly elevated compared to BJWT-infected mice (pb0.05, one-wayANOVA).

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were also significantly enhanced (Fig. 6B). Numbers of neutrophils,NK cells, T cells, CD8+ T cells and B220+ lymphocytes were elevatedin BJ186-infectedmice at day 7 post-infection and CD8+ T cell numberswere ~13-fold higher compared BJWT-infected mice, indicating majordifferences in leukocyte recruitment.

Next, we determined levels of soluble inflammatory mediators inBAL fluids from naïve mice or from mice infected with BJWT or BJ186.Levels of IL-6, MCP-1, IFN-γ and TNF-αwere not significantly differentin BAL from naïve animals compared tomice infected with BJWT at day3 (data not shown) or day 7 post-infection (Fig. 6C). In contrast, levelsof IL-6, MCP-1, TNF-α and IFN-γ in BAL from BJ186-infected mice weresignificantly increased compared to animals infected with BJWT at day7 post-infection.

Vascular leak, pulmonary edema and systemic responses associated withsevere disease in mice infected with BJ186

Vascular leak and pulmonary edema are associated with lungdiseases such as ARDS (Luh and Chiang, 2007) aswell aswith the severedisease observed following neutrophil depletion of influenza virus-infected mice (Tate et al., 2009) or CL-LIP-treatment of BJx109-infectedmice (Tate et al., 2010b). Levels of total protein in BAL supernatantsfrom BJWT or BJ186-infected mice were therefore determined at day 7following infection as a measure of vascular leak. Total protein in BALfrom BJ186-infectedmicewas ~4-fold higher compared to BJWT-infectedmice (Fig. 7A) and the lung wet:dry ratio (Fig. 7B), a measure ofextravascular water (Xing et al., 1993), was also significantly higher forBJ186-infected animals.

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

Systemic manifestations such as leukopenia and thymic atrophyare associated with severe clinical disease during influenza infectionsin humans andmice (Fislova et al., 2009; Peiris et al., 2004; Ryan et al.,2000; Tumpey et al., 2000; Wyde et al., 1977). The severe diseaseobserved in BJ186-infectedmice was associatedwith reduced numbersCD45+ blood leukocytes and circulating neutrophils, B220+ lympho-cytes and CD8+ T cells were significantly reduced compared toanimals infected with BJWT (Fig. 7C). Furthermore, flow cytometricanalysis of thymic cell showed a marked reduction in the number ofdouble positive (DP) thymocytes recovered from BJ186-infected micecompared to animals infected with BJWT (Fig. 7D). Numbers of doublenegative (DN, CD4− CD8−) thymocytes were also reduced in BJ186-infected animals, whereas a modest increase in single positive (SP,CD4+ or CD8+) cells was observed. Immature CD4+ CD8+ DP thy-mocytes are particularly sensitive to glucocorticosteroids inducedas part of the stress response to severe viral infection (Herold et al.,2006). Depletion of DP thymocytes is accompanied by enhancednumbers of SP cells in a number of viral infections (Hayasaka et al.,2007; Onodera et al., 1991), although the mechanisms underlying thisare not clear.

A reverse genetics virus bearing the S186I substitution in the viral HAshows enhanced virulence in mice

To verify that substitution S186I in the viral HAwas responsible forthe enhanced virulence of ppBJ186 viruses, reverse genetics (RG) wasused to generate 6:2 reassortant viruses expressing the HA and NA ofA/Beijing/353/89 (Beij/89; H3N2) and all internal componentsderived from PR8. RG viruses expressing WT Beij/89 HA (RG-BJWT)or Beij/89 HA containing the S186I substitution (RG-BJ186) wererescued, amplified in once in eggs and characterized in vitro and invivo. Sequencing of RNA extracted from allantoic fluid confirmedthat amino acid substitution S186I was retained and that no othersubstitutions had been introduced in HA or NA.

We confirmed that RG-BJ186 (i) agglutinated horse erythrocytesexpressing α(2,3)-Gal-linked Neu5Gc, whereas RG-BJWT did not (datanot shown), and (ii) was less efficient in its ability to infect murine Mϕ(Supplementary Fig. 1A).Moreover, inoculation ofmicewith 105 PFU ofRG-BJWT did not result in any weight loss over a 10-day monitoringperiod, whereas mice infected with an equivalent dose of RG-BJ186 lostweight rapidly and all succumbed to disease (Supplementary Fig. 1B).Together, these data confirm that RG-BJWT and RG-BJ186 behavedsimilarly to BJWT and BJ186 in vitro and in vivo. Thus, amino acid sub-stitution S186I in Beij/89 HA is associated with altered receptorspecificity and virulence in mice.

Discussion

Egg-adapted human H3 viruses show enhanced binding to α(2,3)-Gal-linked SA present on the chicken embryo chorioallantoic mem-brane (CAM), unlike non-adapted counterparts, which show a pref-erence for α(2,6)-Gal-linked SA (Gambaryan et al., 1999; Gambaryanet al., 1997; Ito et al., 1997b). The S186I substitution has been notedfollowing adaptation of H3 subtype influenza viruses to eggs, re-sulting in increased affinity for α(2,3)-Gal-linked-SA (Gambaryanet al., 1999; Gambaryan et al., 1997; Matrosovich et al., 1998). Othersubstitutions, for example G186V, have also been associated withadaptation of H3 viruses to growth in eggs (Lu et al., 2005; Widjajaet al., 2006). Pig serum (PS) is a rich source of α-macroglobulinwhich expresses high levels of α(2,6)-Gal-linked SA (Ryan-Poirierand Kawaoka, 1993) and selection of a PS-resistant mutant ofA/Los Angeles/2/87 (H3N2) was associated with the S186I substitu-tion in HA (Matrosovich et al., 1998; Ryan-Poirier and Kawaoka,1991). Moreover, resistance to the PS inhibitor was associated withpoor recognition of α(2,6)-Gal-linked SA (Gimsa et al., 1996;Matrosovich et al., 1998). In our studies, BJ186 viruses retained the

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Fig. 7. Severe disease during BJ186 infection of B6 mice is associated with pulmonary edema, leukopenia and thymic atrophy. Groups of 5 mice infected with 105 PFU of BJWT or BJ186virus were euthanized and analyzed for pulmonary and systemic manifestations of disease at day 7 post-infection. Naïve mice were included for comparison. (A) Total proteinconcentration in cell-free BAL supernatants. Data shown represent the mean protein concentration±1 SD. (B) Lung wet-to-dry ratios as an assessment of pulmonary edema. Forpanels A/B, *=significantly increased when compared to naïve mice, pb0.05, one-way ANOVA. (C) Viable cell counts were performed on whole blood and flow cytometry was usedto determine numbers of CD45+ inflammatory (inflam) cells, neutrophils (Gr-1high, neut), CD4+ cells (CD4+ TCRβ+), CD8+ T cells (CD8+ TCRβ+) and B220+ cells (B cells).*=numbers from BJ186-infected mice were significantly reduced compared BJWT-infected mice (pb0.05, one-way ANOVA). (D) Viable cell counts were performed on single cellsuspensions prepared from mouse thymi and flow cytometry was used to determine numbers of double negative (CD4− CD8−, DN), double positive (CD4+ CD8+, DP) and singlepositive (CD4+ or CD8+, SP) thymocytes. *=cell numbers significantly elevated or reduced compared to mice infected with BJWT (pb0.05, one-way ANOVA).

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ability to agglutinate erythrocytes expressing α(2,6)-Gal-linkedNeu5Ac but acquired recognition of α(2,3)-Gal-linked Neu5Ac(Table 1).

We propose that the ability of BJ186 viruses to recognize α(2,3)-Gal-linked SA is likely important in enhancing infection of mouseairway epithelial cells and therefore virulence. Compared to BJWT,BJ186 showed increased ability to bind α(2,3)-Gal-linked Neu5Ac(Table 1) and infected LA-4 mouse epithelial cells with greaterefficiency (Fig. 1). Furthermore, BJ186 bound efficiently to LA-4 cells at4 °C but were poor in their ability to elute from the cell surface at 37 °C(Fig. 1C), likely reflecting a disruption in HA/NA balance due toincreased affinity for α(2,3)-Gal-linked SA which is abundantlyexpressed on LA-4 cells (Tate et al., 2011). BJ186 replicated to hightiters in mouse lung following intranasal infection (Fig. 4B), yet lowertiters of BJ186 were detected in cell-free supernatants collected fromLA-4 cells at 16–48 h post-infection (Fig. 1D). In vivo, virus releasedfrom BJ186-infected cells will infect additional epithelial cells, leadingto virus amplification in the lung. In vitro, multiple rounds of virusinfection and amplification will be limited by the number ofsusceptible LA-4 target cells available. Moreover, the enhanced abilityof BJ186 to bind to the surface of LA-4 cells would suggest thatfollowing cytolytic infection, virus releasedmay bind to cellular debrisincluding plasma membranes, and would therefore be removed bycentrifugation prior to plaque assay.

Our previous studies have demonstrated that airwayMϕ expresseshigh levels of α(2,6)-Gal-linked SA compared to α(2,3)-Gal-linked SA(Tate et al., 2011). The S186I substitution of BJ186 was associated withreduced infection of Mϕ; however, BJ186 and BJWT were equallyefficient at agglutinating erythrocytes bearing α(2,6)-Gal-linkedNeu5Ac (Table 1). Our findings would be consistent with the notionthat subtle differences in fine specificity of the receptor-binding site ofBJ186 viruses promote less efficient binding to sialylated speciesexpressed on theMϕ surface. Alternatively, S186Imay disrupt the HA/NA balance to impede efficient attachment to theMϕ surface resultingin less efficient entry and infection via the macrophage mannosereceptor (MMR).

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

BJx109 is sensitive to neutralization by collectins, including SP-D inmouse airway fluids (Reading et al., 1997; Tate et al., in press).Previous studies have shown that resistance to β-inhibitors, includingSP-D, was associatedwith loss of glycosylation site Asn165 from the HAof A/Philippines/2/82 (Anders et al., 1990; Hartley et al., 1992) andincreased virulence in mice (Hartley et al., 1997). Loss of Asn246 fromBeij/89 HA was also associated with resistance to SP-D and enhancedvirulence (Reading et al., 2009) and addition of N-linked glycosyla-tion sites to HA resulted in H3 viruses that were more sensitive toSP-D and less virulent in mice (Vigerust et al., 2007). Residue 186 islocated in the vicinity of the receptor-binding pocket and the S186Imutation does not add or delete an N-linked glycans from HA.Consistent with this, whenmixed with mouse BAL prior to addition toLA-4 cell monolayers, BJWT and BJ186 were neutralized to similar levels(Fig. 3A). However, addition of mouse BAL to LA-4 cells (thereforemimicking the function of pulmonary surfactant in the airways) priorto virus addition showed that BJ186 was more efficient at infectingLA-4 cells in the presence of BAL (Fig. 3B). These results are ofparticular interest when considering the dilute nature of mouse BALcompared to the concentrations of inhibitory proteins present in pul-monary surfactant in vivo. Thus, while BJWT and BJ186 were equivalentin their sensitivity to neutralization by mouse BAL per se (as demon-strated by pre-mixing of each virus with mouse BAL), the enhancedability of BJ186 to bind and infect murine airway epithelial cells mayreduce the effectiveness of SP-D-mediatedneutralization in vivo as BJ186can infect epithelial cells more rapidly, therebyminimizing its exposureto innate immune proteins present in airway fluids.

Infection of mice with BJ186 led to severe disease, characterized byenhanced viral replication in the respiratory tract (Fig. 4B), severepulmonary inflammation (Figs. 5 and 6) and ARDS-like diseaseassociated with vascular leak and pulmonary edema (Fig. 7A/B).Mouse-adapted viruses such as PR8 have a receptor preference forα(2,3)-Gal-linked SA over α(2,6)-Gal-linked SA (Hensley et al., 2009;Suzuki et al., 1986; Tate et al., 2011) and mouse-virulent BJ186 hadacquired the ability to agglutinate erythrocytes expressing α(2,3)-Gal-linked Neu5Ac (Table 1) and horse erythrocytes expressing

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α(2,3)-Gal-linked Neu5Gc (data not shown). Neu5Gc is expressed athigh levels in the lungs of mice (Hedlund et al., 2007; Makatsori et al.,1998; Markotic et al., 1999), suggesting that enhanced binding toα(2,3)-Gal-linked Neu5Ac and/or α(2,3)-Gal-linked Neu5Gc is likelyto contribute to the increased virulence of BJ186. Avian influenzaviruses also display receptor preference for α(2,3)-Gal-linked SA(Connor et al., 1994; Matrosovich et al., 1997) and some strains caninduce a severe ARDS-like disease in mice without prior adaptation(Deng et al., 2010; Xu et al., 2006).

Together, data presented in this study highlight the importance ofreceptor specificity in affecting the ability of influenza virus to interactwith components of innate host defense and in determining diseaseseverity. Previous studies have clearly defined the relationshipbetween glycosylation of HA, sensitivity to collectins and virulencein mice (Reading et al., 1997; Vigerust et al., 2007). Data presentedherein demonstrate that changes in receptor specificity that do notalter HA glycosylation also modulate virulence. During adaptation ofhuman virus strains to growth in mice, a receptor switch to pref-erential recognition of α(2,3)-Gal-linked SA is likely to enhanceinfection of mouse epithelial cells and may also to increase resistanceto innate immune defenses in mouse lung.

Materials and methods

Mice and viruses

C57BL/6 (B6)mice were bred and housed in specific pathogen-freeconditions at the Department of Microbiology and Immunology,University of Melbourne. Male mice 6–8 weeks of age were used in allexperiments. The influenza virus strains A/PR/8/34 Mount Sinai (PR8,H1N1) and BJx109 (H3N2) were obtained from the WHO Collaborat-ing Centre for Reference and Research on Influenza, NorthMelbourne,Australia. BJx109 is a high-yielding reassortants of PR8 with A/Beijing/353/89 (H3N2) that bears that H3N2 surface glycoproteins.

Influenza viruses were grown in 10-day embryonated eggs bystandard procedures and titrated by plaque assay under agarose usingMadin–Darby canine kidney (MDCK) cells (Anders et al., 1994). Dueto differences noted in the plaque size of BJWT and BJ186 viruses (seeResults), titers of infectious virus were confirmed using a single-cyclefluorescent focus assay where monolayers of MDCK cells wereinfected with serial dilutions of each virus. After 1 h, the inoculumwas removed and replaced with serum-free media. At 6–8 h post-infection, cell monolayers were washed and fixed in 80% acetone inwater, and influenza virus-infected cells were detected using mAbclone MP3.1092.IC7 for detection of influenza virus nucleoprotein(NP; WHO Collaborating Centre for Reference and Research onInfluenza, Melbourne, Australia), followed by FITC-conjugated sheepanti-mouse Ig (Silenus, Australia).

Isolation of small plaque mutant of BJx109 from the lungs of mice

Mice were depleted of airway Mϕ via intranasal treatment with100 μl of clodronate-loaded liposomes (CL-LIP; Roche Diagnostics,Germany) while under light anesthesia with isoflurane. Mice weretreated 48 h prior to infection with 105 PFU of BJx109 and every 72h thereafter as described (Tate et al., 2010b). Control mice received anequivalent volume of PBS or saline-loaded liposomes (SL-LIP). At day7 following infection, mice were euthanized; lungs were removed andhomogenised; and titers of infectious virus in clarified homogenateswere determined by plaque assay on MDCK cell monolayers in thepresence of trypsin (Anders et al., 1994). For plaque purification (pp),5 well-separated plaques were picked from 3 different CL-LIP-treatedmice, resuspended in PBS and inoculated into 10-day embryonatedhens' eggs. Allantoic fluid was harvested and frozen at −70 °C.

The genes encoding viral HA and NA glycoproteins were examinedfor nucleotide changes. RNA was extracted from allantoic fluid cell

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

pellet using RNeasy Minikit (Qiagen, USA). Synthesis of cDNA fromRNA served as a template for subsequent PCR reactions preformedwith Qiagen Omniscript RT kit using 20 pmol/μL of Uni12 primer (AGCAAA AGC AGG). DNA encoding the HA and NA genes was amplifiedby PCR using primers as described (Hoffmann et al., 2001). PCRproducts were analyzed by agarose gel electrophoresis and bands ofspecific size were extracted using gel extraction kit (MO BIO, USA).Sequencing was performed by Applied Genetics Diagnostics, Depart-ment of Pathology, The University of Melbourne. H3 numbering(Nobusawa et al., 1991) was used to align the deduced amino acidsequences.

Reassortant influenza viruses used in this study were generated by8-plasmid reverse genetics (RG) as previously described (Hoffmannet al., 2002). Viruses generated were 6:2 reassortants consisting of the6 gene ‘PR8 backbone’ with HA and NA genes from Beij/89. Plasmidvectors (pHW2000) containing the 6 genes of PR8 were kindlyprovided by Dr. Robert Webster, St. Jude Children's Research Hospital,Memphis, TN, USA. A plasmid on the pHW2000 backbone carrying theHA of A/Beijing/353/89 (Beij/89, H3N2) was altered to express S→ I atresidue 186 (H3 numbering) via site-directed mutagenesis usingcloned Pfu DNA polymerase (Stratagene, USA). Parental template wasthen digested via incubation with DpnI (New England Biolabs, USA).Viruses generated were 6:2 reassortants consisting of the PR8 (H1N1)‘backbone’with the HA and NA genes from Beij/89. RG viruses bearingthe HA of Beij/89 (RG-BJWT) or S186I HA (RG-BJ186) were recoveredafter 3 days and amplified in the allantoic cavity of 10-day-oldembryonated hens’ eggs. The HA of RG viruses was sequenced toensure that appropriate sequences were obtained in egg-grown virusstocks.

Desialylation and resialylation of erythrocytes

Methods for the enzymatic modification of erythrocyte oligosac-charides have been described previously (Rogers and Paulson, 1983).In brief, suspensions (10%) human erythrocytes were desialylatedwith 600 mU/ml of C. perfringes sialidase for 1 h at 37 °C, washedtwice and resialylated using 1.5 mM CMP-N-acetylneuraminic acid(NeuAc, Sigma-Aldrich) and 4 mU of either β-galactoside-α2,3-sialyltransferase (α(2,3)-ST, Japan Tobacco Inc., Shizuka, Japan) orβ-D-Galactosyl-β1,4-N-acetyl-β-D-glucosamine-α2,6-sialyltransferase(α(2,6)-ST,Merck, USA) in 100 μl, orwithbuffer alone (sham treated) at37 °C for 4 h. Erythrocytes were washed in TBS and used in standardhemagglutination (HA) titrations.

Virus neutralization assay

To examine the ability of bronchoalveolar lavage (BAL) fluids toneutralize virus infectivity, naïve mice were euthanized and the lungsflushed three times with 1 ml of tris-buffered saline (TBS) through abunted 23-guage needle inserted into the trachea. BAL fluids wereclarified by centrifugation and supernatants frozen at−20 °C. Neutral-ization of influenza virus infectivity was measured by fluorescentfocus reduction in monolayers of LA-4 cells cultured in 96-wellplates as described (Reading et al., 1997). Briefly, BAL supernatantswere diluted in TBS supplemented with 10 mM CaCl2 and incubatedwith a constant amount of virus for 30min prior to the addition to cellmonolayers. In some experiments, dilutions of BAL supernatants wereadded to cell monolayers prior to the addition of virus. Influenza virus-infected cells were detected as described above in the single-cyclefluorescent focus assay. The total number of fluorescent foci in fourrepresentative fields was counted and expressed as a percentage ofthe number of foci in the corresponding area of duplicate controlwells infected with virus alone (i.e. percent of virus control). To testfor inhibition by mannan, diluted BAL supernatant was incubatedwith mannan for 30 min at room temperature prior to the additionof virus.

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Infection of mouse airway Mϕ and epithelial cells

Mouse peritoneal exudate cell (PEC) Mϕ or the murine LA-4 lungepithelial cell line was prepared and infected with virus in 8-wellchamber slides as previously described (Reading et al., 2000). Slideswere stained for expression of influenza virus NP as described aboveandwith 10 μg/ml propidium iodide (Sigma-Aldrich, USA) to visualizenuclei of adherent cells and therefore determine total cell number.The number of fluorescent and total cells was counted in a minimumof four random fields with a minimum of 200 cells counted for eachsample and used to determine the percent of cells infected by virus.To test for inhibition of infection by mannan, cells were incubatedwith mannan (final concentration 10 mg/ml) for 30 min at roomtemperature before addition of virus. In some experiments, LA-4 cellswere infected in chamber slides with 0.01 PFU/cell of virus, washedand cultured in serum-free media in the presence of 4 μg/ml TPCK-treated trypsin. Supernatants were removed at 2, 8, 16, 24 and 48 hpost-infection and clarified by centrifugation and the viral titer (PFU/ml) was determined by plaque assay on MDCK cell monolayers.

Virus binding and elution from epithelial cells

The ability of influenza virus to bind and elute from the surface ofLA-4 cells was performed as described (Reading et al., 2009). Briefly,128 hemagglutinating units (HAU) of influenza virus was added toduplicate tubes, each containing 106 LA-4 cells pre-fixedwith 2% (v/v)PFA. Both samples were incubated on ice for 60 min and then onesamplewas held at 4 °C, while the duplicate samplewas transferred to37 °C for 30 min. A third tube received no cells and was held at 4 °Cthroughout. Samples were centrifuged and virus remaining in cellsupernatant was quantified by standard HA assay and expressed asHAU.

Virus infection of mice

Mice were anesthetized and infected with 105 PFU of influenzavirus via the intranasal (i.n.) route in 50 μl of PBS. Mice were weigheddaily and assessed for visual signs of clinical disease includinginactivity, ruffled fur, laboured breathing and huddling behavior.Animals that had lost ≥25% of their original body weight and/ordisplayed evidence of pneumonia were euthanized. All researchcomplied with the University of Melbourne's Animal ExperimentationEthics guidelines and policies. At various times after infection, micewere euthanized and the lungs and nasal tissues were removed,homogenised in PBS and clarified by centrifugation. Titers ofinfectious virus in tissue homogenates were determined by standardplaque assay on MDCK cells.

Recovery and characterization of leukocytes from mice

BAL cells and heparinized blood were obtained as described (Tateet al., 2008). Samples were treated with Tris–NH4Cl (0.14 M NH4Cl in17 mM Tris, adjusted to pH 7.2) to lyse erythrocytes and washed inRPMI 1640 medium supplemented with 10% FCS (RF10). Cell numbersand cell viability were assessed via trypan blue exclusion.

For flow cytometric analysis, single cell suspensions prepared fromthe blood, BAL and thymus were incubated on ice for 20 min withsupernatants from hybridoma 2.4G2 to block Fc receptors, followed bystaining with appropriate combinations of fluorescein isothiocyanate(FITC)-, phycoerythrin (PE)-, allophycocyanin (APC)-conjugated toGr-1 (RB6-8C5), CD45.2 (104), CD8a (53–6.7), CD4 (GK1.5), B220(RA3-6B2), TCRβ (H57-597) and NK1.1 (PK136). Living cells wereanalyzed by the addition of 10 μg/ml PI to each sample and cells wereanalyzed using a FACSCalibur flow cytometer. A minimum of 50,000live cells (PI−) were collected.

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

Pulmonary histopathology

Lungs were perfused, inflated and fixed in a solution of 4%formaldehyde as previously described (Tate et al., 2009), before 4 μmsections were prepared and stained with hematoxylin and eosin(H&E). Airway inflammation of H&E-stained lung sections was eval-uated on a subjective scale of 0–5 (0=no inflammation, 1=verymild, 2=mild, 3=moderate, 4=marked and 5=severe inflamma-tion) by three independent readers, as described (Tate et al., 2009).Sections were blinded and randomized and samples correspondingto the least severe and most severe inflammation were assignedscores of 0 and 5, respectively. All samples were then graded forperibronchiolar inflammation (around 3–5 small airways per section)and alveolitis in multiple random fields per section by threeindependent readers as described (Sur et al., 1999). Lung sectionswere viewed on a Leica DMI3000 B microscope and photographed at100× magnification unless otherwise stated using a Leica DFC 490camera running from the Leica Application software.

Cytokine bead array for the detection of inflammatory mediators

The levels of IFNγ, TNFα, IL-6, IL-10, IL-12.p70 and MCP-1 in BALsupernatants and serum were determined with the use of a cytokinebead array mouse inflammation kit (Becton Dickinson, USA) accord-ing to the manufacturer's instructions. The detection limit for theassay was 5 pg/mL for all cytokines tested.

Assessment of lung edema and vascular leak

The lung wet-to-dry weight ratio was used as an index of lungwater accumulation during influenza virus infection. Lungs weresurgically dissected, blotted dry and weighed immediately (wetweight). The lung tissue was then dried in an oven at 60 °C for 72 hand re-weighed as dry weight. The ratio of weight-to-dry wascalculated for each animal to assess tissue edema as previouslydescribed (Whitehead et al., 2006). The concentration of protein incell-free BAL supernatant was measured by adding Bradford proteindye. A standard curve using BSA was constructed and the ODdetermined at 595 nm.

Statistical analysis

For the comparison of two sets of values, a Student's t test (two-tailed, two-sample equal variance) was used. When comparing threeor more sets of values, data were analyzed by one-way ANOVA(nonparametric) followed by post-hoc analysis using Tukey'smultiplecomparison test. For the analysis of histopathological data, a Kruskal–Wallis test (nonparametric) was used followed by the Dunn's post-test. Survival proportions were compared using the Mantel Cox logrank test. A p value of ≤0.05 was considered statistically significant.

Supplementarymaterials related to this article can be found onlineat doi: 10.1016/j.virol.2011.01.035.

Acknowledgments

This study was supported by Project Grant #509230 from TheNational Health and Medical Research Council (NH&MRC)of Australia. P.C.R. is a NH&MRC R.D. Wright Research Fellow. TheMelbourne WHO Collaborating Centre for Reference and Research onInfluenza is supported by the Australian Government Department ofHealth and Ageing. The authors wish to thank Dr. Robert Webster,St Jude Children's Research Hospital, Memphis, Tennessee, USA, forprovision of the plasmid vector used to create the reverse engineeredviruses for this study.

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References

Anders, E.M., Hartley, C.A., Jackson, D.C., 1990. Bovine and mouse serum beta inhibitorsof influenza A viruses are mannose-binding lectins. Proc. Natl Acad. Sci. USA 87,4485–4489.

Anders, E.M., Hartley, C.A., Reading, P.C., Ezekowitz, R.A., 1994. Complement-dependentneutralization of influenza virus by a serum mannose-binding lectin. J. Gen. Virol.75 (Pt 3), 615–622.

Baigent, S.J., McCauley, J.W., 2001. Glycosylation of haemagglutinin and stalk-length ofneuraminidase combine to regulate the growth of avian influenza viruses in tissueculture. Virus Res. 79, 177–185.

Briody, B.A., Cassel, W.A., Medill, M.A., 1955. Adaptation of influenza virus to mice. III.Development of resistance to beta-inhibitor. J. Immunol. 74, 41–45.

Brown, E.G., Liu, H., Kit, L.C., Baird, S., Nesrallah, M., 2001. Pattern of mutation in thegenome of influenza A virus on adaptation to increased virulence in the mouselung: identification of functional themes. Proc. Natl Acad. Sci. USA 98, 6883–6888.

Cheung, C.Y., Poon, L.L., Lau, A.S., Luk, W., Lau, Y.L., Shortridge, K.F., Gordon, S., Guan, Y.,Peiris, J.S., 2002. Induction of proinflammatory cytokines in human macrophagesby influenza A (H5N1) viruses: a mechanism for the unusual severity of humandisease? Lancet 360, 1831–1837.

Chu, C.M., 1951. The action of normal mouse serum on influenza virus. J. Gen. Microbiol.5, 739–757.

Conenello, G.M., Zamarin, D., Perrone, L.A., Tumpey, T., Palese, P., 2007. A singlemutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributesto increased virulence. PLoS Pathog. 3, 1414–1421.

Connor, R.J., Kawaoka, Y., Webster, R.G., Paulson, J.C., 1994. Receptor specificity inhuman, avian, and equine H2 and H3 influenza virus isolates. Virology 205, 17–23.

Crouch, E., Wright, J.R., 2001. Surfactant proteins a and d and pulmonary host defense.Annu. Rev. Physiol. 63, 521–554.

Deng, G., Bi, J., Kong, F., Li, X., Xu, Q., Dong, J., Zhang, M., Zhao, L., Luan, Z., Lv, N., Qiao, J.,2010. Acute respiratory distress syndrome induced by H9N2 virus in mice. Arch.Virol. 155, 187–195.

Doms, R.W., Gething, M.J., Henneberry, J., White, J., Helenius, A., 1986. Variant influenzavirus hemagglutinin that induces fusion at elevated pH. J. Virol. 57, 603–613.

Eisen, M.B., Sabesan, S., Skehel, J.J., Wiley, D.C., 1997. Binding of the influenza A virus tocell-surface receptors: structures of five hemagglutinin-sialyloligosaccharidecomplexes determined by X-ray crystallography. Virology 232, 19–31.

Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., 1962. The contribution of sialic acid tothe surface charge of the erythrocyte. J. Biol. Chem. 237, 1992–2000.

Fislova, T., Gocnik, M., Sladkova, T., Durmanova, V., Rajcani, J., Vareckova, E., Mucha, V.,Kostolansky, F., 2009. Multiorgan distribution of human influenza A virus strainsobserved in a mouse model. Arch. Virol. 154, 409–419.

Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H.D., Stech, J., 2005. The viralpolymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc. Natl Acad. Sci. USA 102, 18590–18595.

Gambaryan, A.S., Tuzikov, A.B., Piskarev, V.E., Yamnikova, S.S., Lvov, D.K., Robertson, J.S.,Bovin, N.V., Matrosovich, M.N., 1997. Specification of receptor-binding phenotypesof influenza virus isolates from different hosts using synthetic sialylglycopolymers:non-egg-adapted human H1 and H3 influenza A and influenza B viruses share acommon high binding affinity for 6′-sialyl(N-acetyllactosamine). Virology 232,345–350.

Gambaryan, A.S., Robertson, J.S., Matrosovich, M.N., 1999. Effects of egg-adaptation onthe receptor-binding properties of human influenza A and B viruses. Virology 258,232–239.

Gao, P., Watanabe, S., Ito, T., Goto, H., Wells, K., McGregor, M., Cooley, A.J., Kawaoka, Y.,1999. Biological heterogeneity, including systemic replication in mice, of H5N1influenza A virus isolates from humans in Hong Kong. J. Virol. 73, 3184–3189.

Gimsa, U., Grotzinger, I., Gimsa, J., 1996. Two evolutionary strategies of influenzaviruses to escape host non-specific inhibitors: alteration of hemagglutinin orneuraminidase specificity. Virus Res. 42, 127–135.

Glaser, L., Conenello, G., Paulson, J., Palese, P., 2007. Effective replication of humaninfluenza viruses in mice lacking a major alpha2, 6 sialyltransferase. Virus Res. 126,9–18.

Hartley, C.A., Jackson, D.C., Anders, E.M., 1992. Two distinct serum mannose-bindinglectins function as beta inhibitors of influenza virus: identification of bovine serumbeta inhibitor as conglutinin. J. Virol. 66, 4358–4363.

Hartley, C.A., Reading, P.C., Ward, A.C., Anders, E.M., 1997. Changes in thehemagglutinin molecule of influenza type A (H3N2) virus associated withincreased virulence for mice. Arch. Virol. 142, 75–88.

Hatta, M., Gao, P., Halfmann, P., Kawaoka, Y., 2001. Molecular basis for high virulence ofHong Kong H5N1 influenza A viruses. Science 293, 1840–1842.

Hawgood, S., Brown, C., Edmondson, J., Stumbaugh, A., Allen, L., Goerke, J., Clark, H.,Poulain, F., 2004. Pulmonary collectins modulate strain-specific influenza a virusinfection and host responses. J. Virol. 78, 8565–8572.

Hayasaka, D., Ennis, F.A., Terajima, M., 2007. Pathogeneses of respiratory infections withvirulent and attenuated vaccinia viruses. Virol. J. 4, 22.

Hedlund, M., Tangvoranuntakul, P., Takematsu, H., Long, J.M., Housley, G.D., Kozutsumi,Y., Suzuki, A., Wynshaw-Boris, A., Ryan, A.F., Gallo, R.L., Varki, N., Varki, A., 2007. N-glycolylneuraminic acid deficiency in mice: implications for human biology andevolution. Mol. Cell. Biol. 27, 4340–4346.

Hensley, S.E., Das, S.R., Bailey, A.L., Schmidt, L.M., Hickman, H.D., Jayaraman, A.,Viswanathan, K., Raman, R., Sasisekharan, R., Bennink, J.R., Yewdell, J.W., 2009.Hemagglutinin receptor binding avidity drives influenza A virus antigenic drift.Science 326, 734–736.

Herold, M.J., McPherson, K.G., Reichardt, H.M., 2006. Glucocorticoids in T cell apoptosisand function. Cell. Mol. Life Sci. 63, 60–72.

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

Hoffmann, E., Krauss, S., Perez, D., Webby, R., Webster, R.G., 2002. Eight-plasmid systemfor rapid generation of influenza virus vaccines. Vaccine 20, 3165–3170.

Hofmann, P., Sprenger, H., Kaufmann, A., Bender, A., Hasse, C., Nain, M., Gemsa, D., 1997.Susceptibility of mononuclear phagocytes to influenza A virus infection andpossible role in the antiviral response. J. Leukoc. Biol. 61, 408–414.

Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set forthe full-length amplification of all influenza A viruses. Arch. Virol. 146, 2275–2289.

Ito, T., Couceiro, J.N., Kelm, S., Baum, L.G., Krauss, S., Castrucci, M.R., Donatelli, I., Kida, H.,Paulson, J.C., Webster, R.G., Kawaoka, Y., 1998. Molecular basis for the generation inpigs of influenza A viruses with pandemic potential. J. Virol. 72, 7367–7373.

Ito, T., Suzuki, Y., Mitnaul, L., Vines, A., Kida, H., Kawaoka, Y., 1997a. Receptor specificityof influenza A viruses correlates with the agglutination of erythrocytes fromdifferent animal species. Virology 227, 493–499.

Ito, T., Suzuki, Y., Takada, A., Kawamoto, A., Otsuki, K., Masuda, H., Yamada, M., Suzuki,T., Kida, H., Kawaoka, Y., 1997b. Differences in sialic acid-galactose linkages in thechicken egg amnion and allantois influence human influenza virus receptorspecificity and variant selection. J. Virol. 71, 3357–3362.

Keleta, L., Ibricevic, A., Bovin, N.V., Brody, S.L., Brown, E.G., 2008. Experimental evolutionof human influenza virus H3 hemagglutinin in the mouse lung identifies adaptiveregions in HA1 and HA2. J. Virol. 82, 11599–11608.

Lu, B., Zhou, H., Ye, D., Kemble, G., Jin, H., 2005. Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing thehemagglutinin and neuraminidase activities, using reverse genetics. J. Virol. 79,6763–6771.

Luh, S.P., Chiang, C.H., 2007. Acute lung injury/acute respiratory distress syndrome(ALI/ARDS): the mechanism, present strategies and future perspectives oftherapies. J. Zhejiang Univ. Sci. B 8, 60–69.

Makatsori, E., Fermani, K., Aletras, A., Karamanos, N.K., Tsegenidis, T., 1998. Screening ofN-acylneuraminic acids in serum and tissue specimens of mouse C57BI with Lewis'lung cancer by high-performance liquid chromatography. J. Chromatogr. B Biomed.Sci. Appl. 712, 23–29.

Markotic, A., Lumen, R., Marusic, A., Jonjic, S., Muthing, J., 1999. Ganglioside expressionin tissues of mice lacking the tumor necrosis factor receptor 1. Carbohydr. Res. 321,75–87.

Matrosovich, M.N., Gambaryan, A.S., Teneberg, S., Piskarev, V.E., Yamnikova, S.S., Lvov,D.K., Robertson, J.S., Karlsson, K.A., 1997. Avian influenza A viruses differ fromhuman viruses by recognition of sialyloligosaccharides and gangliosides and by ahigher conservation of the HA receptor-binding site. Virology 233, 224–234.

Matrosovich, M., Gao, P., Kawaoka, Y., 1998. Molecular mechanisms of serum resistanceof human influenza H3N2 virus and their involvement in virus adaptation in a newhost. J. Virol. 72, 6373–6380.

Matrosovich, M.N., Matrosovich, T.Y., Gray, T., Roberts, N.A., Klenk, H.D., 2004. Humanand avian influenza viruses target different cell types in cultures of human airwayepithelium. Proc. Natl Acad. Sci. USA 101, 4620–4624.

McCullers, J.A., Hoffmann, E., Huber, V.C., Nickerson, A.D., 2005. A single amino acidchange in the C-terminal domain of the matrix protein M1 of influenza B virusconfers mouse adaptation and virulence. Virology 336, 318–326.

Medeiros, R., Escriou, N., Naffakh, N., Manuguerra, J.C., van der Werf, S., 2001.Hemagglutinin residues of recent human A(H3N2) influenza viruses thatcontribute to the inability to agglutinate chicken erythrocytes. Virology 289, 74–85.

Medeiros, R., Naffakh, N., Manuguerra, J.C., van der Werf, S., 2004. Binding of thehemagglutinin from human or equine influenza H3 viruses to the receptor isaltered by substitutions at residue 193. Arch. Virol. 149, 1663–1671.

Ning, Z.Y., Luo, M.Y., Qi, W.B., Yu, B., Jiao, P.R., Liao, M., 2009. Detection of expression ofinfluenza virus receptors in tissues of BALB/c mice by histochemistry. Vet. Res.Commun. 33, 895–903.

Nobusawa, E., Aoyama, T., Kato, H., Suzuki, Y., Tateno, Y., Nakajima, K., 1991. Comparisonof complete amino acid sequences and receptor-binding properties among 13serotypes of hemagglutinins of influenza A viruses. Virology 182, 475–485.

Nobusawa, E., Ishihara, H., Morishita, T., Sato, K., Nakajima, K., 2000. Change in receptor-binding specificity of recent human influenza A viruses (H3N2): a single amino acidchange in hemagglutinin altered its recognition of sialyloligosaccharides. Virology278, 587–596.

Onodera, T., Taniguchi, T., Tsuda, T., Yoshihara, K., Shimizu, S., Sato, M., Awaya, A., Hayashi,T., 1991. Thymic atrophy in type 2 reovirus infected mice: immunosuppression andeffects of thymic hormone. Thymic atrophy caused by reo-2. Thymus 18, 95–109.

Peiris, J.S., Yu, W.C., Leung, C.W., Cheung, C.Y., Ng, W.F., Nicholls, J.M., Ng, T.K., Chan,K.H., Lai, S.T., Lim, W.L., Yuen, K.Y., Guan, Y., 2004. Re-emergence of fatal humaninfluenza A subtype H5N1 disease. Lancet 363, 617–619.

Pekosz, A., Newby, C., Bose, P.S., Lutz, A., 2009. Sialic acid recognition is a keydeterminant of influenza A virus tropism in murine trachea epithelial cell cultures.Virology 386, 61–67.

Perrone, L.A., Plowden, J.K., Garcia-Sastre, A., Katz, J.M., Tumpey, T.M., 2008. H5N1 and1918 pandemic influenza virus infection results in early and excessive infiltrationof macrophages and neutrophils in the lungs of mice. PLoS Pathog. 4, e1000115.

Pillai, S.P., Lee, C.W., 2010. Species and age related differences in the type anddistribution of influenza virus receptors in different tissues of chickens, ducks andturkeys. Virol. J. 7, 5.

Reading, P.C., Morey, L.S., Crouch, E.C., Anders, E.M., 1997. Collectin-mediated antiviralhost defense of the lung: evidence from influenza virus infection of mice. J. Virol.71, 8204–8212.

Reading, P.C., Miller, J.L., Anders, E.M., 2000. Involvement of the mannose receptor ininfection of macrophages by influenza virus. J. Virol. 74, 5190–5197.

Reading, P.C., Pickett, D.L., Tate, M.D., Whitney, P.G., Job, E.R., Brooks, A.G., 2009. Loss ofa single N-linked glycan from the hemagglutinin of influenza virus is associatedwith resistance to collectins and increased virulence in mice. Respir. Res. 10, 117.

e influenza virus hemagglutinin modulates sensitivity to soluble6/j.virol.2011.01.035

11M.D. Tate et al. / Virology xxx (2011) xxx–xxx

Reading, P.C., Whitney, P.G., Pickett, D.L., Tate, M.D., Brooks, A.G., 2010. Influenzaviruses differ in ability to infect macrophages and to induce a local inflammatoryresponse following intraperitoneal injection of mice. Immunol. Cell Biol. 88,641–650.

Rodgers, B., Mims, C.A., 1981. Interaction of influenza virus with mouse macrophages.Infect. Immun. 31, 751–757.

Rogers, G.N., Paulson, J.C., 1983. Receptor determinants of human and animal influenzavirus isolates: differences in receptor specificity of the H3 hemagglutinin based onspecies of origin. Virology 127, 361–373.

Russell, R.J., Stevens, D.J., Haire, L.F., Gamblin, S.J., Skehel, J.J., 2006. Avian and humanreceptor binding by hemagglutinins of influenza A viruses. Glycoconj. J. 23, 85–92.

Ryan, L.K., Neldon, D.L., Bishop, L.R., Gilmour, M.I., Daniels, M.J., Sailstad, D.M., Selgrade,M.J., 2000. Exposure to ultraviolet radiation enhances mortality and pathologyassociated with influenza virus infection in mice. Photochem. Photobiol. 72,497–507.

Ryan-Poirier, K.A., Kawaoka, Y., 1991. Distinct glycoprotein inhibitors of influenza Avirus in different animal sera. J. Virol. 65, 389–395.

Ryan-Poirier, K.A., Kawaoka, Y., 1993. Alpha 2-macroglobulin is the major neutralizinginhibitor of influenza A virus in pig serum. Virology 193, 974–976.

Sauter, N.K., Hanson, J.E., Glick, G.D., Brown, J.H., Crowther, R.L., Park, S.J., Skehel, J.J.,Wiley, D.C., 1992. Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonancespectroscopy and X-ray crystallography. Biochemistry 31, 9609–9621.

Shilov, A.A., Sinitsyn, B.V., 1994. Changes in its hemagglutinin during the adaptation ofthe influenza virus to mice and their role in the acquisition of virulent propertiesand resistance to serum inhibitors. Vopr. Virusol. 39, 153–157.

Skehel, J.J., Wiley, D.C., 2000. Receptor binding andmembrane fusion in virus entry: theinfluenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569.

Smeenk, C.A., Wright, K.E., Burns, B.F., Thaker, A.J., Brown, E.G., 1996. Mutations in thehemagglutinin and matrix genes of a virulent influenza virus variant, A/FM/1/47-MA, control different stages in pathogenesis. Virus Res. 44, 79–95.

Stoner, G.D., Kikkawa, Y., Kniazeff, A.J., Miyai, K., Wagner, R.M., 1975. Clonal isolation ofepithelial cells from mouse lung adenoma. Cancer Res. 35, 2177–2185.

Sur, S., Wild, J.S., Choudhury, B.K., Sur, N., Alam, R., Klinman, D.M., 1999. Long termprevention of allergic lung inflammation in a mouse model of asthma by CpGoligodeoxynucleotides. J. Immunol. 162, 6284–6293.

Suzuki, Y., Matsunaga, M., Matsumoto, M., 1985. N-Acetylneuraminyllactosylceramide,GM3-NeuAc, a new influenza A virus receptor which mediates the adsorption-fusion process of viral infection. Binding specificity of influenza virus A/Aichi/2/68(H3N2) to membrane-associated GM3 with different molecular species of sialicacid. J. Biol. Chem. 260, 1362–1365.

Suzuki, Y., Nagao, Y., Kato, H., Matsumoto, M., Nerome, K., Nakajima, K., Nobusawa, E.,1986. Human influenza A virus hemagglutinin distinguishes sialyloligosaccharidesin membrane-associated gangliosides as its receptor which mediates theadsorption and fusion processes of virus infection. Specificity for oligosaccharidesand sialic acids and the sequence to which sialic acid is attached. J. Biol. Chem. 261,17057–17061.

Suzuki, T., Takahashi, T., Guo, C.T., Hidari, K.I., Miyamoto, D., Goto, H., Kawaoka, Y.,Suzuki, Y., 2005. Sialidase activity of influenza A virus in an endocytic pathwayenhances viral replication. J. Virol. 79, 11705–11715.

Sweet, C., Smith, H., 1980. Pathogenicity of influenza virus. Microbiol. Rev. 44, 303–330.

Please cite this article as: Tate, M.D., et al., Receptor specificity of thcollectins of the innate immune system ..., Virology (2011), doi:10.101

Tate, M.D., Brooks, A.G., Reading, P.C., 2008. The role of neutrophils in the upper andlower respiratory tract during influenza virus infection of mice. Respir. Res. 9, 57.

Tate, M.D., Deng, Y.M., Jones, J.E., Anderson, G.P., Brooks, A.G., Reading, P.C., 2009.Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection. J. Immunol. 183, 7441–7450.

Tate, M.D., Brooks, A.G., Reading, P.C., 2010a. Inhibition of lectin-mediated innate hostdefences in vivo modulates disease severity during influenza virus infection.Immunol Cell Bio. In press.

Tate, M.D., Pickett, D.L., van Rooijen, N., Brooks, A.G., Reading, P.C., 2010b. Critical role ofairway macrophages in modulating disease severity during influenza virusinfection of mice. J. Virol. 84, 7569–7580.

Tate, M.D., Brooks, A.G., Reading, P.C., 2011. Correlation between sialic acid expressionand infection of murinemacrophages by different strains of influenza virus. Microb.Infect. 13, 202–207.

Tumpey, T.M., Garcia-Sastre, A., Taubenberger, J.K., Palese, P., Swayne, D.E., Pantin-Jackwood, M.J., Schultz-Cherry, S., Solorzano, A., Van Rooijen, N., Katz, J.M., Basler,C.F., 2005. Pathogenicity of influenza viruses with genes from the 1918 pandemicvirus: functional roles of alveolar macrophages and neutrophils in limiting virusreplication and mortality in mice. J. Virol. 79, 14933–14944.

Tumpey, T.M., Lu, X., Morken, T., Zaki, S.R., Katz, J.M., 2000. Depletion of lymphocytesand diminished cytokine production in mice infected with a highly virulentinfluenza A (H5N1) virus isolated from humans. J. Virol. 74, 6105–6116.

Vigerust, D.J., Ulett, K.B., Boyd, K.L., Madsen, J., Hawgood, S., McCullers, J.A., 2007. N-linked glycosylation attenuates H3N2 influenza viruses. J. Virol. 81, 8593–8600.

Vines, A., Wells, K., Matrosovich, M., Castrucci, M.R., Ito, T., Kawaoka, Y., 1998. The roleof influenza A virus hemagglutinin residues 226 and 228 in receptor specificity andhost range restriction. J. Virol. 72, 7626–7631.

Wagner, R., Matrosovich, M., Klenk, H.D., 2002. Functional balance betweenhaemagglutinin and neuraminidase in influenza virus infections. Rev. Med. Virol.12, 159–166.

Weis, W., Brown, J.H., Cusack, S., Paulson, J.C., Skehel, J.J., Wiley, D.C., 1988. Structure ofthe influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature333, 426–431.

Wells, M.A., Albrecht, P., Daniel, S., Ennis, F.A., 1978. Host defense mechanisms againstinfluenza virus: interaction of influenza virus with murine macrophages in vitro.Infect. Immun. 22, 758–762.

Whitehead, G.S., Burch, L.H., Berman, K.G., Piantadosi, C.A., Schwartz, D.A., 2006.Genetic basis of murine responses to hyperoxia-induced lung injury. Immunoge-netics 58, 793–804.

Widjaja, L., Ilyushina, N., Webster, R.G., Webby, R.J., 2006. Molecular changes associatedwith adaptation of human influenza A virus in embryonated chicken eggs. Virology350, 137–145.

Wyde, P.R., Couch, R.B., Mackler, B.F., Cate, T.R., Levy, B.M., 1977. Effects of low- andhigh-passage influenza virus infection in normal and nude mice. Infect. Immun. 15,221–229.

Xing, Z., Kirpalani, H., Torry, D., Jordana, M., Gauldie, J., 1993. Polymorphonuclearleukocytes as a significant source of tumor necrosis factor-alpha in endotoxin-challenged lung tissue. Am. J. Pathol. 143, 1009–1015.

Xu, T., Qiao, J., Zhao, L., Wang, G., He, G., Li, K., Tian, Y., Gao, M., Wang, J., Wang, H., Dong,C., 2006. Acute respiratory distress syndrome induced by avian influenza A (H5N1)virus in mice. Am. J. Respir. Crit. Care Med. 174, 1011–1017.

e influenza virus hemagglutinin modulates sensitivity to soluble6/j.virol.2011.01.035