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Contents lists available at ScienceDirect

Vaccine

jou rn al hom ep age: www.elsev ier .com/ locat e/vacc ine

ow pathogenic avian influenza A(H7N9) virus causes high mortalityn ferrets upon intratracheal challenge: A model to studyntervention strategies

.H.C.M. Kreijtza, E.J.B. Veldhuis Kroezeb, K.J. Stittelaarb, L. de Waalb, G. van Amerongenb,. van Trieruma, P. van Runa, T. Bestebroera, T.Kuikena, R.A.M. Fouchiera,.F. Rimmelzwaana,b, A.D.M.E. Osterhausa,b,∗

Department of Viroscience, Erasmus MC, Rotterdam, The NetherlandsViroclinics Biosciences, Rotterdam, The Netherlands

r t i c l e i n f o

rticle history:eceived 24 May 2013ccepted 20 June 2013vailable online xxx

eywords:7N9vian influenza A

ntervention strategies

a b s t r a c t

Infections with low pathogenic avian influenza (LPAI) A(H7N9) viruses have caused more than 100 hos-pitalized human cases of severe influenza in China since February 2013 with a case fatality rate exceeding25%. Most of these human infections presented with severe viral pneumonia, while limited informationis available currently on the occurrence of mild and subclinical cases. In the present study, a ferret modelfor this virus infection in humans is presented to evaluate the pathogenesis of the infection in a mam-malian host, as ferrets have been shown to mimic the pathogenesis of human infection with influenzaviruses most closely. Ferrets were inoculated intratracheally with increasing doses (>10 e5 TCID50) ofH7N9 influenza virus A/Anhui/1/2013 and were monitored for clinical and virological parameters up to

four days post infection. Virus replication was detected in the upper and lower respiratory tracts whileanimals developed fatal viral pneumonia. This study illustrates the high pathogenicity of LPAI-H7N9 virusfor mammals. Furthermore, the intratracheal inoculation route in ferrets proofs to offer a solid model forLPAI-H7N9 virus induced pneumonia in humans. This model will facilitate the development and assess-ment of clinical intervention strategies for LPAI-H7N9 virus infection in humans, such as preventivevaccination and the use of antivirals.

. Introduction

Avian influenza viruses have crossed the species barrier on sev-ral occasions and with varying impact on human health. Fromebruary 2013 onward several human cases with severe respiratoryllness were reported from South-East China. The causative agents

ere rapidly characterized and subtyped as low pathogenic aviannfluenza (LPAI) A(H7N9) viruses that most likely were the resultf multiple reassortment events of at least two avian influenzairuses [1,2]. Although the exact source of the human infectionsith this apparently avian influenza virus remains to be elucidated,

equence analysis has indicated that the viruses have been circu-

Please cite this article in press as: Kreijtz JHCM, et al. Low pathogenic aintratracheal challenge: A model to study intervention strategies. Vaccine

ating for a longer period before they recently surfaced [3,4], Sincehe first reported human cases, the virus has infected at least 131umans of which 32 succumbed to the infection with a predilection

∗ Corresponding author at: Department of Viroscience, Erasmus MC, PO Box 2040,000 CA Rotterdam, The Netherlands. Tel.: +31 10 7044066; fax: +31 10 7044760.

E-mail addresses: a.osterhaus@erasmusmc.nl,va.editorialassistant.vaccine@gmail.com (A.D.M.E. Osterhaus).

264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.vaccine.2013.06.071

© 2013 Elsevier Ltd. All rights reserved.

for older male individuals [5–8]. It is the largest outbreak of avianinfluenza in humans since the introduction of highly pathogenicavian influenza (HPAI) A(H5N1) viruses in the human populationthat thus far has resulted in over 600 reported cases with a hos-pitalized case fatality rate of approximately 60%. The majority ofthe humans infected with LPAI-H7N9 virus have presented withsevere viral pneumonia and became critically ill [2]. The virus hasbeen classified as being low pathogenic based on the genotype(the hemagglutinin does not contain a multi-basic cleavage site)and based on the results of intravenous pathogenicity index (IVPI)testing in chickens and data from poultry and wild birds. How-ever, the clinical manifestation of LPAI-H7N9 virus infection inhumans shows a higher pathogenic phenotype. To further explorethis discrepancy and elucidate the pathogenesis of the infection inmammals, we established a model for LPAI-H7N9 virus-inducedpneumonia in ferrets (Mustela Putorius furo). The ferret model hasbeen used to elucidate the severity of lower respiratory tract infec-

vian influenza A(H7N9) virus causes high mortality in ferrets upon(2013), http://dx.doi.org/10.1016/j.vaccine.2013.06.071

tions with various influenza A virus subtypes [9]. Preliminary dataon intranasal inoculation of ferrets with avian influenza A(H7N9)indicates that the virus causes only mild disease (unpublished data)in these animals. However, we have demonstrated in the past that

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he choice of inoculation route has a major impact on the outcomend severity of influenza virus infection in ferrets [10]. Since theast majority of currently recorded human cases of LPAI-H7N9 virusnfection have been characterized by severe viral pneumonia, repli-ation in the lower respiratory route seems to be the major causef at least severe human disease. Therefore intratrachael admin-stration of the virus to ferrets would be the route of choice totudy the pathogenesis of severe LPAI-H7N9 virus infection in thisammalian model.

. Materials and methods

.1. Virus

Influenza virus A/Anhui/1/2013 (H7N9) isolated from a fataluman case in China was kindly provided by the Pandemic

nfluenza Preparedness (PIP) Framework. The virus had been iso-ated and passaged three times in embryonated chicken eggs and

as subsequently passaged once in Madin Darby Canine Kid-ey (MDCK) cells. The infectious virus titer was determined asescribed previously and expressed in tissue culture infectious dose0% (TCID50) [11].

.2. Animals

Healthy outbred female ferrets (Mustela Putorius furo), of around2 months of age and seronegative for antibodies against aleu-ian disease virus and seasonal influenza viruses were used. Aboutwo months prior to the start of the experiment, the animalsere anesthetized using a cocktail of ketamine (Alfasan, Woer-en, The Netherlands) and domitor (Orion Pharma, Espoo, Finland),nd a temperature logger (DST milli-T logger; Star-Oddi, Reyk-avik, Iceland) was placed in their peritoneal cavity to record theody temperature every 10 min. The animals were maintained intandard housing, and provided with commercial food pellets andater ad libitum and were placed in BSL-3 isolator units just before

noculation. Ferrets were inoculated intratracheally with influenzairus A/Anhui/1/2013 (H7N9) at a dose of 105 (n = 2), 106 (n = 3),07 (n = 2) or 108 (n = 2) TCID50 in a volume of 3 ml. An indepen-ent animal ethics committee approved the experimental protocolefore the start of the experiments.

.3. Virus replication in the upper and lower respiratory tract

Nasal and pharyngeal swabs were taken daily during the infec-ion period. After spontaneous death or euthanasia (see Section 3)hree or four days after inoculation, samples of all lobes of theight lung and the accessory lobe, nasal turbinates, tonsils, tra-hea, bronchus, tracheobronchial lymph nodes and lungs wereollected and snap frozen using a dry ice/ethanol bath and storedt −70 ◦C until further processing. Tissue samples were weighednd subsequently homogenized with the FastPrep-24 (MP Biomed-cals, Eindhoven, The Netherlands) in Hank’s balanced salt solutionontaining 0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin,00 �g/ml streptomycin, 100 U/ml polymyxin B sulfate, 250 �g/mlentamycin, and 50 U/ml nystatin (ICN Pharmaceuticals, Zoeter-eer, The Netherlands) and centrifuged briefly before dilution.uadruplicate 10-fold serial dilutions of lung and swab super-atants were used to determine the presence of viral RNA byaqman and infectious virus titers in confluent layers of MDCK cellss described previously [11].

Please cite this article in press as: Kreijtz JHCM, et al. Low pathogenic aintratracheal challenge: A model to study intervention strategies. Vaccine

.4. Histopathological examination and immunohistochemistry

At necropsy all ferrets (n = 9) and their organs were grosslyxamined by opening the thoracic, abdominal, and cranial

PRESSe xxx (2013) xxx– xxx

cavities. The extent of pulmonary consolidation was assessed basedon visual estimation of the percentage of affected lung tissue. Therelative lung weight was calculated as proportion of the bodyweight (lung weight/body weight × 100). The left lung was rou-tinely collected for histological examination, by means of cutting4 standard sections per animal (one cross section and one sagittalsection from the cranial lobe, and one cross section and one sagi-ttal section from the caudal lobe). Besides the lungs other organsthat were sampled for histology included: nasal turbinates, brainsincluding olfactory bulb, tonsil, trachea, tracheobronchial lymphn-odes, heart, liver, stomach, small and large intestines, pancreas,spleen, adrenal, kidney, urinary bladder, ovaries, uterus, skele-tal muscle (quadriceps), femoral bone marrow. All tissues wereimmersed in 10% neutral-buffered formalin for fixation, routinelyprocessed, paraffin embedded, cut to 4 �m on glass slides andstained with hematoxylin and eosin (H&E) for histopathologicalevaluation. Serial sections of the respiratory tract and brains werestained for influenza A nucleoprotein (NP) by immunohistochem-istry (IHC) as described previously [12].

3. Results

3.1. Clinical signs

All infected animals developed fever with a mean peak bodytemperature of 41.2 ◦C (SD = 0.53) within 24–48 h post infec-tion. From two days post inoculation (dpi) onwards the animalsdeveloped signs of respiratory distress (presented as heavy breath-ing/dyspnoe) and eventually hunched posture. During the course ofinfection the animals’ food and water intake decreased, resultingin mild emaciation and dehydration. On day 3 post infection thetwo animals inoculated with 108 TCID50 and animals inoculatedwith 107 (n = 1) and 106 (n = 1) TCID50 succumbed. At day 3 postinfection the surviving animals were lethargic and by day 4 oneadditional animal (106 TCID50) had succumbed whereas one ani-mal (107 TCID50) was moribund, Necropsies were performed on allthe deceased animals and the surviving animals after euthanasia.

3.2. Virus replication in the respiratory tract

Pharyngeal swabs from all animals tested positive for virus withthe highest virus titers 3 dpi in those obtained from ferrets inoc-ulated with 107 or 108 TCID50: 104.4 TCID50/ml (SD = 100.1–101.3).Virus replication in the ferrets inoculated with 105 or 106 TCID50reached peak values 4 dpi (Fig. 1A). These data confirmed the detec-tion of viral RNA in the throat by real tie PCR. In seven out of nineanimals viral RNA was detected in the nose and this again was con-firmed by virus isolation with the peak of virus replication on day3 post infection: 103.6 TCID50/ml (SD = 100.4) in the ferrets inocu-lated with 108 TCID50 (Fig. 1B). On day 4 rectal swabs were takenfrom the remaining animals and in 40% of the animals viral RNAwas detected but no infectious virus could detected.

All animals tested positive for virus replication in nasalturbinates, tonsils, trachea, bronchus, bronchial lymph nodes andlungs (Fig. 1C). In the bronchial lymph nodes and lungs the meanvirus titers were highest (105.7–106.8 and 105.0–1055.8 TCID50/g,respectively). Only in the trachea the virus titers were higher for fer-rets inoculated with 108 TCID50 i: 106.6 TCID50/gr (SD = 101.3). Apartfrom the bronchial lymph nodes and lungs, virus titers were highestin the samples of the ferrets inoculated with 107 and 108 TCID50,illustrating a dose dependency of the LPAI-H7N9 virus infection.

vian influenza A(H7N9) virus causes high mortality in ferrets upon(2013), http://dx.doi.org/10.1016/j.vaccine.2013.06.071

3.3. Histopathological changes and immunohistochemistry

In all the nine animals the foremost macroscopic post-mortemlesions concerned the lungs. The lung lesions ranged from extensive

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Fig. 1. Virus replication in the respiratory tract. After inoculation with avian influenza A(H7N9) virus, pharyngeal (A) and nasal (B) swabs were taken daily to monitor virusr fferenT comb

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eplication at these sites over time. Upon necropsy, samples were taken from the dio calculate the mean virus titers of the respiratory tract samples (C) the data were

ultifocal (Fig. 2A) to almost diffuse dark red pulmonary consolida-ion (Fig. 2D) with edematous frothy fluid oozing from the primaryronchi upon section. The most severely affected lungs additionallyisplayed a pale mottled aspect, likely due to trapped air. The extentf pulmonary consolidation was assessed based on visual estima-ion of the percentage of affected lung area from the pleural aspect.ll animals were found to have practically empty gastrointestinal

racts combined with a subtle pale reticular pattern of the livers,ndicative of hepatic lipidosis due to inappetence. Except for thewo lowest dosed animals, all other spleens were slight to moder-tely swollen and hyperemic. No further macroscopic lesions werencountered (Table 1).

On histopathological examination, the nasal turbinates of theerrets inoculated with the two highest dosages (n = 4: 107 and 108

CID ) were moderately inflamed. These rhinitides were char-

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50cterized by moderate numbers of mainly neutrophils infiltratedithin the nasal respiratory epithelium and underlying Lamina pro-

ria. These lesions co-localized with epithelial cells positive for

ig. 2. Representative (histo)pathological changes of the lungs. From left to right, ventrtained with H&E (B and E) and immunohistochemistry (IHC) (C and F) for Influenza A vntratracheally inoculated with 105 TCID50 Avian influenza A(H7N9) virus, that was grosnflamed bronchiole containing an intraluminal plug of neutrophils and cellular debrisroteinaceous material. The bottom panel depicts animal#8 inoculated similarly with 108

ikewise depicting a bronchiole on the right and adjoining alveoli, but more severely inflameHC stains show the influenza virus infected epithelial cells by dark reddish-brown staine

t locations of the respiratory tract for the detection of replication competent virus.ined from the two (105, 107, 108 TCID50) or three (106 TCID50) animals per group.

influenza A virus nucleoprotein (NP) by immunohistochemistry(IHC). The nasal olfactory epithelium was typically not or verymildly affected. None of the other ferrets displayed rhinitis, andthe nasal turbinates were negative for influenza A virus NP byIHC (Table 2). The severity of inflammatory lesions in the tracheaswas relatively mild, with lesions composed of few neutrophils andlesser lymphocytes within the Lamina propria. These occurred inthree animals inoculated with the three highest virus doses. Theserelatively mild lesions co-localized with tracheal epithelial cellspositive for influenza A virus NP by IHC, however several tra-cheas were positive for NP without showing cellular inflammatoryreaction. In the bronchi of all animals mild to moderate lesionswere present that were mainly limited to epithelial necrosis ofthe submucosal glands, mostly without affecting the luminal lin-ing epithelium. The severity and extent of this necrotizing bronchial

vian influenza A(H7N9) virus causes high mortality in ferrets upon(2013), http://dx.doi.org/10.1016/j.vaccine.2013.06.071

submucosal adenitis was associated and co-localized with the num-ber of glandular cells positive for influenza A virus NP by IHC. Themost severe cases of bronchial submucosal adenitis were found

al viewed gross lung lesions (A and D), corresponding microscopic lesions seriallyirus Nucleoprotein (NP), respectively. The top panel depicts animal#1 which wassly affected for approximately 50%. The photomicrographs depict on the right an, the adjoining inflamed alveoli contain neutrophils and macrophages and someTCID50, which was grossly affected for approximately 90%, with photomicrographsd with necrosis and flooding by intense eosinophilic edema fluid. The correspondingd nuclei. (Original microscopic magnifications of 400×).

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Table 1Clinical parameters.

Nr Virus dose (TCID50) Day of death Weight loss (%) Body temperature

Peak (◦C) Time post inoculation (h)

1 105 4a 11.2 40.8 332 4a 10.9 41.3 31

3 106 4b 12.4 41.7 244 3b 10.3 41.0 245 4a 14.4 41.3 24

6 107 3b 9.8 40.1 217 4c 14.0 41.8 23

8 108 3b 7.4 41.2 249 3b 8.8 41.8 16

a Sacrificed end experiment.b Found dead.c Euthanized moribund.

Table 2Lung parameters & Immunohistochemistry.

Nr Virus dose (TCID50) Relative lung weighta Affected lung (%) Immunohistochemistry of respiratory tract epitheliumb

Nasal cavity Trachea Bronchi Bronchioles Alveoli

Glands Lining

1 105 2.08 50 − + + + + ++2 1.21 30 − + + + ++ ++

3 106 2.52 50 − + ++ + + ++4 1.74 90 − ++ + + + ++5 1.70 50 − − + + ++ ++

6 107 3.94 90 + + + + ++ ++7 3.87 90 + − + + + ++

8 108 2.99 90 ++ + ++ + + ++9 2.28 90 ++ − ++ + ++ ++

HC: −

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a Relative lung weight = (lung weight/body weight) × 100%.b Semiquantative parameter for number of influenza A virus NP positive cells in I

n the ferrets inoculated with the two highest virus concentra-ions. All animals showed inflammatory bronchiolar lesions. These

arked to severe bronchiolitides were characterized by intraep-thelial infiltrates of neutrophils mainly, with in the most severeases plugging of bronchioli by sloughed necrotic epithelial cellsdmixed with mucus containing degenerated neutrophils. In theost severely affected animals nearly all bronchioles were com-

letely denuded. Rather the extent, than the severity of affectedronchioles was associated with virus concentrations inoculated.he necrotizing bronchiolitis co-localized with the number ofpithelial cells strongly positive for influenza A virus NP by IHC.owever, many severely affected bronchioles were completelyenuded of their epithelium with only exfoliated cellular debrisositive for NP.

The pattern of inflammation and/or alveolar damage was com-arable between all animals, whereas the severity and extent varied

n relation to virus doses inoculated. All animals suffered fromn acute necrotizing (broncho)interstitial pneumonia whilst theeverity combined with the extent ranged from moderate (Fig. 2Bnd C) to severe (Fig. 2E and F). Affected alveoli were centered morer less around inflamed bronchioles in the moderate cases, andere coalescing into extensive affected areas in the more severe

ases. Within the affected alveoli the septa were only slightly thick-ned with some edema fluid and infiltrated neutrophils, and lesseracrophages and lymphocytes, without noticeable type II pneu-

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ocyte hyperplasia. The lining pneumocytes were mostly necroticnd exfoliated in the affected alveolar areas. In the moderate caseshe outlines of the affected alveolar septa were present, whereas inhe most severe cases the alveolar septa were completely necrotic

= none, + = some, ++ = many.

and their outlines poorly discernable or sometimes even collapsed.The alveolar lumina were markedly flooded by protein rich edemafluid containing neutrophils, macrophages, erythrocytes, degener-ated exfoliated cells or cellular debris, and many fibrin strands.These lesions within the alveoli co-localized with lining and exfoli-ated pneumocytes strongly positive for influenza A virus NP by IHC.All of the animals’ brains including olfactory bulbs were negativefor influenza A virus NP by IHC, and histopathology confirmed theslight hepatic lipidosis in all animals. The animals displayed someestrous activity but no bone marrow depression as a result of thatand no further abnormalities were observed.

4. Discussion

Despite the low pathogenic classification of LPAI-H7N9 virus forpoultry, it does cause high morbidity and mortality in ferrets exper-imentally infected by the intratracheal route. All animals developedclinical signs of severe disease caused by severe viral pneumonia. Ahigher infectious dose correlated with earlier onset of disease anda more severe outcome and ultimately death within the time frameof the study.

Virus replication was detected both in the upper and lowerrespiratory tract of all animals in this study, which can be explainedby the receptor specificity of the H7 hemagglutinin. The receptorbinding pocket mutation Q226L that is found in this virus is asso-

vian influenza A(H7N9) virus causes high mortality in ferrets upon(2013), http://dx.doi.org/10.1016/j.vaccine.2013.06.071

ciated with a shift in receptor preference of the virus from �2,3to �2,6-linked sialic acids [2]. The exact receptor specificity of theavian influenza A(H7N9) virus has to be determined but it seemsthat the virus can bind to both �2,3 to �2,6-linked sialic acids. The

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atter are predominant in the upper respiratory tract, thus correlat-ng with the rhinitis found in the animals inoculated with 107 and08 TCID50 (n = 4). Most likely, due to the high viral load in thesenimals, the virus was able to spread more easily from the phar-nx to the nasal cavity and was able to replicate there, resulting innflammation of the nasal turbinates.

Besides replication in the upper respiratory tract the virus waslso detected in the lower respiratory tract, most likely associatedith the �2,3-linked sialic acids that are found primarily deep In

he lung, thus explaining the severe viral pneumonia observed here.onclusively the LPAI-H7N9 virus can infect cells both in the uppernd lower respiratory tract, similar to pH1N1 influenza virus [9].PAI-H5N1 viruses on the contrary primarily replicate in the lower

espiratory tract and are able to spread to the central nervous sys-em and other solid organs. In the current study the LPAI-H7N9irus did not spread outside the context of the respiratory tract.owever the fact that within the respiratory tract the virus could

pread to the upper region has implications for the risk assessmentf the virus. The receptor distribution and binding of human andvian influenza viruses in the respiratory tract of the ferret is sim-lar to that of humans. And since the virus is capable of spreadingpwards through the respiratory tract this increases the risk of virusxcretion which could lead to transmission [13–15].

In the N9 neuraminidase of the LPAI-H7N9 virus a deletion wasetected. The lack of these five specific amino acids has been asso-iated with enhanced virus replication and alteration of the virusropism [2]. Apart from receptor specificity, the avian influenza(H7N9) virus has additional pathogenicity markers. The L89V andspecially the E627K mutation, associated with enhanced poly-erase activity and enhanced virulence in the context of avian

nfluenza A(H5N1) virus, are present in the polymerase protein PB2f the influenza virus A/Anhui/1/2013 [2]. The presence of thesearkers may account for the severe viral pneumonia that the virus

auses in humans and in the ferret model described here.This study illustrates the potential of the ‘low pathogenic’ LPAI-

7N9 virus to infect the lower respiratory tract of mammalsnd cause severe viral pneumonia in a manner that is compara-le to HPAI-H5N1 virus. This raises questions about whether theathogenicity classification of avian influenza viruses should notlso take into account the pathogenicity phenotype of the virus inammals. Taken together the data presented in this paper illustrate

hat intratracheal LPAI-H7N9 virus infection of ferrets, like othernfections with different influenza virus subtypes, can be used tonvestigate the pathogenesis of this infection in mammals, as wells urgently needed intervention strategies like vaccination and these of antivirals.

Please cite this article in press as: Kreijtz JHCM, et al. Low pathogenic aintratracheal challenge: A model to study intervention strategies. Vaccine

cknowledgements

The authors would like to thank the Pandemic Influenzareparedness (PIP) Network and Dr. Shu (CDC, China) and

[

PRESSe xxx (2013) xxx– xxx 5

Dr. J. McCauley (Mill Hill, Londen, UK) for providing the avianinfluenza A(H7N9) virus. D. van Riel, W. van Aert, R. Boom, S.Berkhof and P. Nuijten provided excellent technical assistance.JK and AO are sponsored by ERC Grant Fluplan 250136 and ERCGrant ARCAS 324634. RF is sponsored by NIAID-NIH contractHHSN266200700010C. PR and TK are sponsored by FP7 contract278976 ANTIGONE.

Conflict of interest: The authors KS, EVK and LdW are fulltimeemployed by Erasmus MC spin-off company ViroClinics BioSciencesB.V. GvA, GR and AO are part-time employed by ViroClinics Bio-Sciences B.V. of which AO is chief scientific officer.

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