Using the Ferret as an Animal Model for Investigating Influenza Antiviral Effectiveness

12 Pages·2016·1.13 MB·English

REVIEW
published:04February2016
doi:10.3389/fmicb.2016.00080
Using the Ferret as an Animal Model
for Investigating Influenza Antiviral
Effectiveness
DingY.Oh1,2*andAeronC.Hurt1,3*
1WHOCollaboratingCentreforReferenceandResearchonInfluenza,VictorianInfectiousDiseasesReferenceLaboratory,
PeterDohertyInstituteforInfectionandImmunity,Melbourne,VIC,Australia,2SchoolofAppliedandBiomedicalSciences,
FederationUniversityAustralia,Gippsland,VIC,Australia,3MelbourneSchoolofPopulationandGlobalHealth,Universityof
Melbourne,Parkville,VIC,Australia
The concern of the emergence of a pandemic influenza virus has sparked an
increasedefforttowardthedevelopmentandtestingofnovelinfluenzaantivirals.Central
to this is the animal model of influenza infection, which has played an important
role in understanding treatment effectiveness and the effect of antivirals on host
immune responses. Among the different animal models of influenza, ferrets can be
consideredthemostsuitableforantiviralstudiesastheydisplaymostofthehuman-like
Editedby: symptomsfollowinginfluenzainfections,theycanbeinfectedwithhumaninfluenzavirus
SlobodanPaessler, without prior viral adaptation and have the ability to transmit influenza virus efficiently
UniversityofTexasMedicalBranch,
between one another. However, an accurate assessment of the effectiveness of an
USA
Reviewedby: antiviral treatment in ferrets is dependent on three major experimental considerations
PaulHorwood, encompassing firstly, the volume and titer of virus, and the route of viral inoculation.
InstitutPasteurinCambodia,
Secondly, the route and dose of drug administration, and lastly, the different methods
Cambodia
BarryRockx, used to assess clinical symptoms, viral shedding kinetics and host immune responses
RijksinstituutvoorVolksgezondheiden in the ferrets. A good understanding of these areas is necessary to achieve data that
Milieu,Netherlands
can accurately inform the human use of influenza antivirals. In this review, we discuss
*Correspondence:
the current progress and the challenges faced in these three major areas when using
DingY.Oh
dingthomas.oh@influenzacentre.org; theferretmodeltomeasureinfluenzaantiviraleffectiveness.
AeronC.Hurt
aeron.hurt@influenzacentre.org Keywords:ferret,antiviral,animalmodel,influenza,effectiveness
Specialtysection:
Thisarticlewassubmittedto INFLUENZA – THE DISEASE
Virology,
asectionofthejournal Influenza is a highly contagious respiratory disease causing symptoms ranging from headache,
FrontiersinMicrobiology
myalgia,malaise,sorethroat,sneezing,andnasaldischarge(CoxandSubbarao,1999).Influenza
Received:02December2015 virusistransmittedviavirus-ladensecretionspropelledbycoughingorsneezingfromaninfected
Accepted:18January2016
person. Most influenza infections are self-limiting, lasting for one to 5 days but host factors
Published:04February2016
such as age, pregnancy, and underlying medical conditions can increase the severity of illness
Citation:
(Gavin and Thomson, 2003). Influenza causes high global mortality and morbidity annually,
OhDY andHurtAC(2016)Using
withUnitedStatesaloneexperiencingapproximately95,000–172,000hospitalizationsand21,000–
theFerretasanAnimalModel
41,000deathsannually(Lowenetal.,2006).Themorbidityassociatedwithseasonalinfluenzahasa
forInvestigatingInfluenzaAntiviral
significanteconomicimpactduetoworkabsenteeismandputshugepressureonthepublichealth
Effectiveness.Front.Microbiol.7:80.
doi:10.3389/fmicb.2016.00080 system.
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OhandHurt AntiviralEffectivenessUsingtheFerret
INFLUENZA ANTIVIRALS andnewtherapeutics(Kisoetal.,2010;MargineandKrammer,
2014; Marjuki et al., 2014). To date, there are many different
To date, the neuraminidase inhibitors (NAIs) are the only animalmodelsofinfluenzainfection,namelyferrets,mice,guinea
licensed class of antiviral drugs effective against currently pigs, swine, non-human primates (NHP), and more recently,
circulating influenza viruses. Zanamivir (RelenzaTM) and zebrafish(Gaboretal.,2014).Theprosandconsofthedifferent
oseltamivir (TamifluTM) have been licensed since 1999 while animal models of influenza to investigate disease pathogenesis,
newer NAIs, such as peramivir (RapivabTM) and laninamivir transmission,andvaccinedevelopmenthavebeenwell-described
(InavirTM),areapproved inJapan,and inthe case ofperamivir inseveralpublishedreviewsandaresummarizedhereinTable1
also in South Korea, USA, and China (Ikematsu and Kawai, (Bouvier and Lowen, 2010; Lowen et al., 2014; Margine and
2011;Chairat et al.,2013; ChinaBioToday, 2013). However,the Krammer,2014;ThangavelandBouvier,2014;Davisetal.,2015;
therapeutic and prophylactic efficacy of NAIs against ‘seasonal’ EnkirchandvonMessling,2015).
influenzainfectionremainshotlydebated(Jeffersonetal.,2014).
The continuous risks posed by the emergence of NAI-resistant Animal Models in Influenza Antiviral
viruses (Takashita et al., 2015) and the pandemic potential of Studies
avianinfluenza viruses, such as A(H5N1) (Nguyen et al., 2013)
Among all animal experimental models, mice are most
and A(H7N9) (Hu et al., 2013), has sparked a major effort to
commonly used for testing influenza antivirals mainly due to
developnewantiviralsforhumanuse.
factors,suchaslowerexperimentalcost,easeofanimalhandling
Typically, investigationalantiviralswillfirst undergoinvitro
andtheabilitytouselargenumbersofanimalstoattainstatistical
efficacy screening, followed by in vivotesting in animalmodels
power in a single experiment (Ryan et al., 1994; Mendel et al.,
tolook atpharmacokinetics/pharmacodynamics (PK/PD), drug
1998; Triana-Baltzer et al., 2009; Kiso et al., 2010; Bantia et al.,
toxicityanddrugeffectivenesspriortoclinicaltrials.Assuch,the
2011; Smee et al., 2012b; Zarogiannis et al., 2012; Marjuki
choiceoftheanimalmodelforassessingtheeffectivenessofthese
et al., 2014). To date, weight loss, mortality (lethal model) and
influenza antivirals becomes critical as it provides pre-clinical
virus titer are the commonly used determinants of antiviral
datathatcaninformthedecisionforprogressiontowardclinical
drugeffectivenessinmicestudies.Althoughthesemeasurements
trials.Currently,therearealargenumberofinfluenzaantivirals
are informative, the usefulness of mice in antiviral studies has
undergoingclinicaltrials,asubstantialincreasefromthelimited
been largely limited by the lack of clinical symptoms following
trialsin2000(Figure1).Inthemajorityofhumanclinicaltrialsof
influenza infection. The absence of clinical symptoms such as
influenzaantivirals,theprimaryendpointusedtoassessthedrug
fever,sneezing,nasaldischarge,andnasalinflammationinmice
efficacy is the time to alleviation of clinical symptoms, such as
following influenza infection limits the extrapolation of mouse
cough,fever,sorethroat,myalgia,lethargy,nasalcongestion,and
data to the human scenario where alleviation of symptoms are
headaches,whereasotheraspects,includingtheabilitytoreduce
considered as the primary endpoint in clinical trials (Table 1).
viral shedding, are considered secondary endpoints (Hayden
In contrast, the ferret is the only animal model which displays
etal.,1997;TheMIST,1998;Makelaetal.,2000;Nicholsonetal.,
comparable clinical symptoms to that of humans following
2000;Treanoretal.,2000;Haffizullaetal.,2014).
influenza infection (Table 1). In view of these factors, in this
reviewwe will discussthe current progress, limitations and the
ANIMAL MODELS IN INFLUENZA future directions ofusingferretstoassessantiviraleffectiveness
RESEARCH againstinfluenzainfections.
Ferret
Animalmodelsofinfluenza infection haveplayedanimportant
roleintheunderstandingofviralpathogenicityandhaveserved Since the discovery of the susceptibility of ferrets (Mustela
as pre-clinical models for the evaluation of vaccine candidates putorius furo) to influenza virus in the 1930’s (Smith et al.,
FIGURE1|Overviewofclinicaltrialsofinfluenzaantiviralsinyear2000and2015.Datafor2015extractedfromclinicaltrials.gov(ClinicalTrials,2015)using
searchterms:‘Influenza’and‘antivirals’and‘antiviralstreatment’.
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OhandHurt AntiviralEffectivenessUsingtheFerret
TABLE1|Comparisonofdifferentanimalmodelsforinfluenzainfection.
Clinicalsymptoms Human Animalmodelofinfluenzainfection
Ferrets Mice GuineaPigs Swine NHP Zebrafish
Sneezing Yes Yes No No Yes Notalwaysa No
Nasaldischarge Yes Yes No No Notalwaysa Notalwaysa No
Lethargy Yes Yes Yes No No Notalwaysa Unknown
Fever Yes Yes No No Notalwaysa Notalwaysa No
Weightloss Yes Yes Yes No Minor Notalwaysa No
Viralshedding Yes Yes Yes Yes Yes Yes Yes
Experimentalcost − Moderate Low Moderate High High Low
Transmissionbetweenanimals − Good Poor Good Good Poor Unknown
Caninfectwithhumaninfluenzaviruses? − Yes Nob Yes Yes Yes Unknown
A(H1N1)pdm09c − Yes Yes Yes Yes Yes Notreported
A(H3N2)c − Yes Yes Yes Yes Yes Yes
Bc - Yes Yes Notreported Yes Yes Notreported
Avianoriginc − Yes Yes Yes Notreported Yes Notreported
NHP,Non-humanprimates.aClinicalsymptomsvarywithdifferentinfluenzastrains.bSerialpassagingisrequiredto‘adapt’thevirustoreplicateinmajorityofmouse
strains.cInfluenzavirusstrain/subtypethathavebeentestedintheanimalmodel.
1933), they remain one of the best animal models of influenza regarding the husbandry of ferrets for animal experimentation.
infectionastheyexhibitmanyoftheclinicalsymptomsobserved Therefore in the absence of any local directives, animal ethics
inhumansfollowinginfluenzainfection,canbedirectlyinfected committeesmayrelyonguidelinesprovidedbyothercountries,
with human influenza virus without prior viral adaptation, particularlywherethecommitteehaslittleexperienceintheuse
and have the ability to transmit influenza virus efficiently offerrets.
between one another (Table 1). The susceptibility of ferrets Despite these limitations, the use of ferrets in influenza
to human influenza viruses is due to the presence of α2- research has increased considerably since 2008 (Figure 2).
6-linked terminal N-acetylneuraminic sialic acids (Neu5AC) This increment was largely attributed to a global effort to
in their respiratory tract which facilitates virus binding and better understand viruses with pandemic potential such as the
the initiation of viral replication (Jia et al., 2014; Ng et al., avian influenza viruses A(H5N1) and A(H7N9), and the virus
2014). responsible for the 2009 pandemic, the A(H1N1)pdm09 virus
However, the use of ferrets for influenza studies has (Govorkova etal.,2005;Yenetal.,2007;Boltzetal.,2008;Itoh
been limited by factors such as animal availability, genetic etal.,2009;Mainesetal.,2009;Munsteretal.,2009;Belseretal.,
heterogeneity (out-bred) (Margine and Krammer, 2014), the 2013a;Richard etal.,2013;Zhangetal.,2013).With the recent
requirementofacomplex husbandryfacility andcagingsystem publication of the ferret genome (Peng et al., 2014), it is likely
(Mabry et al., 2013; Enkirch and von Messling, 2015), and a thattheuseoftheferretasamodelofinfluenzawillcontinueto
lackofimmunologicalreagentsandgeneticallymodifiedmutants rise.
for immunological investigation (Margine and Krammer, 2014;
Enkirch and von Messling, 2015). As a consequence, ferret
experiments can be limited by small sample sizes (n ≤ 5)
(Belser et al., 2013b; Nishiura et al., 2013; Buhnerkempe et al.,
2015), where large animal-to-animal variability has resulted in
the detection of non-statistically significant trends of antiviral
effectivenessbetweenthe treatmentgroups invariables,suchas
weight, temperature, nasal inflammation, and virus titer (Rowe
et al., 2010; Govorkova et al., 2011; Oh et al., 2014, 2015).
Ideally,alargernumberofferretsshouldbeusedbutlimitations,
such as high experimental cost, low animal availability, limited
caging capacity and ethical constraint, typically restricts most
studies to group sizes of five or less ferrets. Unlike the more
commonlyusedanimalmodels,suchasrodentsandguineapigs,
the use of larger animals for experimentation, such as ferrets, FIGURE2|Thenumberofpublicationsusingdifferentanimalmodels
can be met with greater scrutiny by animal ethics governing ofinfluenza.Numberofpaperspublishedontopicsrelatingtoinfluenzaand
bodiesonaspects,suchasthechoiceofanimal,animalnumbers mice/ferret/guineapigfrom1950to2013.Datatabulatedbyonlineautomated
yearlystatisticsofPubMedresults(http://dan.corlan.net/medline-trend.html).
and husbandry concerns. Only a small number of countries
Searchtermsusedare‘Influenza’and‘Mice/ferret/guineapig.’
or regions (such as USA, UK, and Europe) have guidelines
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OhandHurt AntiviralEffectivenessUsingtheFerret
To date, the ferret model has been used to investigate viral INOCULATION OF INFLUENZA VIRUS IN
susceptibility and transmission (Yen et al., 2007; Maines et al., FERRETS
2009;Zhangetal.,2013),andasapreclinicalmodeltoinvestigate
poorly understood areas such as immunological responses to The initiation of influenza infection in animal experimental
influenza in young children (newly weaned ferrets) (Huang models,andeveninthe humanchallengemodel(Darton etal.,
et al., 2012), impaired immunity in older individuals (aged 2015),isroutinelycarriedoutbyinstillingvirusviatheintranasal
ferrets) (Paquette et al., 2014) and the treatment effectiveness route. In general, intranasal instillation of virus is considered
of different antivirals to reduce influenza infection (Boltz etal., to be a simple procedure (Turner et al., 2011) compared to
2008;Kuboetal.,2010;Govorkovaetal.,2011;Kitanoetal.,2011; intratracheal (Kreijtz et al., 2013; van der Vries et al., 2013)
Marriott et al., 2014). Recently, the use of ferrets in influenza or ocular (Belser et al., 2014), by which ferrets can also be
antiviral studies has extended to treatment effectiveness in an infected in a reproducible manner. However, the decision on
immunocompromised setting (van der Vries et al., 2013), the which inoculation route to initiate infection can be dependent
effectivenessofantiviraltreatmentorprophylaxis inpreventing onthevirusstraintobetested.Foravianinfluenzaviruses,such
infections in secondary contacts (Oh et al., 2014) and the asA(H5N1)andA(H7N9),intratrachealinstillationispreferred
transmissionofinfluenzabetweenaninfantandmotherduring overdirectinoculationintothelowerrespiratorytractofferrets,
breast-feeding(Paquetteetal.,2015). as it is found to induce a more severe pneumonia that more
There are a large number of experimental variables in a closelyresemblespathogenesisobservedinhumans,comparedto
typicalferretantiviraleffectivenessstudythatcanalterthestudy intranasalinoculationwhichcausesmildtomoderatepneumonia
outcome andthatshould be carefullyconsidered toensure that inferrets(Bodewesetal.,2011;Kreijtzetal.,2013).Forhuman
themostreliabledataisgenerated.Thesevariablesarediscussed seasonal viruses, such as influenza A(H1N1)pdm09, A(H3N2),
indetailbelowandaresummarizedinFigure3. and B viruses, intratrachel instillation has been used in only a
FIGURE3|Overviewofthethreeexperimentalconsiderationswhenassessinganantiviralinaferretmodelofinfluenzainfection.i.n:Intranasal;i.p,
intraperitoneal;i.v,intravenous;i.t,intratracheal;PK/PD,pharmacokinetics/pharmacodynamics;BAL,bronchoalveolarlavage;AUC,areaundercurve.
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OhandHurt AntiviralEffectivenessUsingtheFerret
small number of studies (van der Vries et al., 2013). Instead,
ferretscanbeeffectivelyinfectedbyintranasalinstillation(Huang
etal.,2011).However,theclinicalseverityofinfluenzainfection
via intranasal instillation is highly dependent on the volume of
inoculum (Bodewes et al., 2011; Moore et al., 2014). A larger
volume of inoculum (1 mL) results in the delivery of virus
down into lower respiratory tract and into the lungs compared
to smaller volumes (0.2 mL and 0.5 mL) which are primarily
retained in the upper respiratory tract (Moore et al., 2014). As
a result, ferrets intranasally infected with the larger volumes of
inoculum have a more severe illness and lung histopathology
compared to those infected with a smaller volume (0.2 mL)
even when the same infectious titer of virus was used (Moore
etal.,2014).Thesefindingnotonlyunderscoredinfluenzastrains,
inoculum route and volume to be important considerations in
the ferret model of influenza infection but also highlighted the
difficulties in interpreting data across studies using different
inoculationprotocols. FIGURE4|Schematicrepresentationoftheviralsheddingkineticsof
Thealternativestointranasalinfectionaretheuseofa‘natural’ intranasallyinfected(102–103,104–106TCID50viralinoculum)ferrets,
ferretsnaturallyinfectedbycontacttransmissionviaaninfected
infection, thatcan be achievedbyeither contact (Roberts etal.,
donorandintranasallyinfectedhumaninachallengemodel(Hayden
2012; Oh et al., 2014) or non-contact transmission (Hamelin etal.,1999;Baccametal.,2006).Ferretviraldatawereadaptedfrom
et al., 2011; van der Vries et al., 2011; Herfst et al., 2012) of publishedstudies(McBrayeretal.,2010;Smithetal.,2011;Starketal.,
virus by exposing a naïve ferret to an infected ferret in the 2013;Ohetal.,2014,2015).
same or adjacent cage, or aerosolized challenge (Gustin et al.,
2011).Despite‘naturalinfection’beingamoreclinicallyrelevant
route of transmission, few studies have used this methodology, short time period (Maines et al., 2009; Govorkova et al., 2011;
presumably due to the additional experimental time involved, Kimetal.,2013;Starketal.,2013;Paquetteetal.,2014;Marjuki
additional ferret use, the requirement for larger cages to house et al., 2015). Ferrets infected with high viral inocula (104−6
multiple animals for contact transmission or a more complex TCID /animal) can show viral shedding kinetics where titers
50
caging system for non-contact transmission, and the lack of peak as early as 1 day post-infection (Figure 4). In contrast,
transmissibility of influenza viruses, such as avian influenza ferretsinfectedwithalowerviralinocula(102−3TCID /animal)
50
A(H5N1)andA(H7N9)(Yenetal.,2007;Belseretal.,2013a)and canshowamore‘typical’viralsheddingcurvethatmoreclosely
influenzaBvirus(Kimetal.,2015),viatheaerosoland/orcontact resembles a natural infection where viral titers gradually rise,
route. Nevertheless, ‘natural’ infection has the clear benefit of peakandthenfall(Baccametal.,2006;Figure4).Althoughhigh
infecting ferretswith arealistic infectious viraldoseand results titer viral inoculums have been used to increase the chances of
in viral replication kinetics that better mimic those of ‘natural’ infection or increase pathogenicity, several studies have shown
influenza infections in humans (Figure 4). Unlike ‘natural’ thattheinitialtiteroftheinoculumdoesnotcorrelatetodisease
infection, which relies on infected donor ferrets to transmit severityinferrets(Marriottetal.,2014;Ohetal.,2015).
viruses to naïve recipients, a more standardized alternative is Influenzaantivirals,suchasNAIs,typicallyactbyinterrupting
aerosolchallengewhichinvolvesexposingnaïveferretsdirectlyto theviralreplication cycle,whereasantibiotics directly eliminate
knowndosesofaerosolizedinfluenzavirusesdeliveredbyspecific and reduce the causative pathogen (McCullers, 2011).
devices. Ferrets exposed to low aerosolized viral dose (1.5–2.2 Therefore, the common practice of infecting ferrets with a
FID )havebeenshowntoexhibitsimilarviralsheddingkinetics high viral inoculum could overwhelm the host (ferret) with
50
anddiseasepathogenesistothoseinfectedvia‘natural’infection an unrealistically large number of infectious viral particles and
(Gustin et al., 2011). In contrast to ‘natural’ infection, aerosol under such experimental conditions, the effectiveness of an
challenges eliminates the need for donor ferrets, allows ferrets antiviraltreatmentcouldbeunderminedastheantiviralswould
to be infected with viruses with poor aerosol transmissibility, not be able to contain such rapid onset of viral infection. As
and enables the manipulation of variables, such as dose and demonstrated by Marriott et al. (2014), oseltamivir treatment
timingofviralexposure,toachieveamorestandardizedwayof significantly lowered viral shedding, lowered inflammatory
infectingferretsina‘natural’fashion.However,fewstudieshave nasal cell count and improved activity levels following a 102
reportedusingthismethodologypresumablyduetothehighcost PFU/animal dose of infection, but had no significant effect on
involved in requiring specialized aerosol system and cages for theseparameterswhenahighviralinoculumof106PFU/animal
suchinoculationprotocol. was used (Marriott et al., 2014). Therefore, in the context
The majority of ferret infection studies have used intranasal of antiviral testing in ferrets and in particular to intranasal
infections with inocula of high infectious viral titers (e.g., 106 inoculation,carefulconsiderationshouldbegiventotheamount
TCID /PFU/EID peranimal)whichmayresultinaverylarge of virus used, to prevent undermining the effectiveness of
50 50
numberof infectious particles infecting the nasalepithelial ina antiviralsininfluenzainfection.
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OhandHurt AntiviralEffectivenessUsingtheFerret
DRUG ADMINISTRATION zanamivir and laninamivir are delivered to humans as a dry
powder formulation that is actively inhaled by the patient via
Administrationoftherapeuticsintoanimalsrequiresthecareful speciallydesignedinhalers(Chairatetal.,2013).Mimickingthis
considerationofmanyfactorsincludingthedrugpharmacology, typeofadministrationinanimalsisdifficult,andispresumably
concentration, volume, timing, frequency of dose and route of why the majority of animal studies investigating zanamivir
administration (Urso et al., 2002). The antivirals for influenza (Herlocheretal.,2003;Hurtetal.,2010;Smeeetal.,2012a,b)and
treatment that are currently approved or in late-phase clinical laninamivir (Kubo et al., 2010) have dissolved the compounds
trials in humans encompass various routes of administration in saline or water and administered intranasally (Table 2).
includingoral,inhaledandintravenous(Table2).Wherepossible While intranasal instillation of drugsis asimple technique that
the route of administration in animals should follow the same can be easily adopted by most laboratories, the relevance of
route of delivery as in humans so that the results can be delivering the drug in this manner when it is designed to be
better extrapolated to the findings in man (Turner et al., 2011; inhaled is questionable (Turner et al., 2011). In addition, it
Table2). has been demonstrated in mice that intranasal instillation of
The delivery of parenterally administered antivirals, such as drug administration can exacerbate viral infection leading to
peramivir, in ferrets is relatively straight-forward and can be lower drug effectiveness (Smee et al., 2012b). Insufflators are
easily administered by intravenous injection (Yun et al., 2008; devicesthathavebeenwidelyusedasanon-invasivemethodto
Kitano et al., 2011). Administering drugs via the enteral (oral) administerpowdereddrugsfordrugdepositionstudiesinsmall
route in ferrets can be limited by many factors such as the animalssuchasmiceorrats(Naharetal.,2013),buthavebeen
requirement for multiple dosing regimens, poor drug solubility lesscommonlyusedinlargeranimalssuchasferrets.Tobridge
andpalatabilityofthedrugtoanimals.Inferrets,directfeeding this unmet need for a delivery system for powdered antivirals
oforallyadministeredcompounds ispreferred overoralgavage such as laninamivir, we recently characterized the usage of a
(which is commonly used in mice) as it does not require drypowderinsufflator todeliverlaninamivir octanoate (LO)to
anesthesia,whichcanimpactontheanimal’shealthandbehavior ferretspriortoinfluenzainfection(Panozzoetal.,2015).Invitro
ifgivenrepeatedly,suchastwicedailyfor5days.Foroseltamivir laserdiffractionanalysisshowedthat∼80%LOtogetherwithits
administration,ferretscanbefedoseltamivirphosphatedissolved lactosecarriercanbeeffectivelydischargedfromthedeviceand
in sugar solution to achieve an accurate dosing (Oh et al., intratrachealadministrationofLOinanesthetizedferretscanbe
2014, 2015). The drug in sugar solution is well-tolerated by easily performed (Panozzo et al., 2015). For LO where a single
ferrets allowing multiple dosing without the need for sedation, dose is sufficient, the method is highly applicable, but repeated
althoughhigherdosesofthedrug(e.g.,25mg/kg)arelessreadily dosingwouldbedifficultduetotheneedforregularanesthesia.
swallowed than the standard 5 mg/kg dose. In contrast, oral The use of the insufflator device may be useful to investigate
antivirals with poor solubility, such as nitazoxanide (Haffizulla otherinhaleddrugsinferrets,suchasDAS181.
etal.,2014;Rossignol,2014),posedifficultieswhenadministering
toferrets,andthereforealternativemethods,suchasspikingthe
drug into food or in special ‘treats’, may be necessary (Turner DRUG DOSAGE
etal.,2011).
The delivery of inhaled drugs, such as zanamivir and Besides route of administration, accurate evaluation of the
laninamivir, to ferrets poses additional challenges. Both effectiveness of antivirals in animals is also dependent on the
TABLE2|Thedifferencesinantiviraldrugadministrationroutebetweenhumanandanimalmodelofinfluenzainfection.
Drugs Approvedadministration Typicaladministrationrouteusedinanimalstudies Reference
routeinhuman
Ferrets Mice
Amantadine Oral Intraperitoneal Oral Herlocheretal.,2003;Bantiaetal.,2011;Smee
etal.,2012a
Rimantadine Oral NR Oral Smeeetal.,2012a
Oseltamivir Oral Oral Oral Govorkovaetal.,2011;Marriottetal.,2014;Oh
etal.,2014,2015;Birdetal.,2015;Maroisetal.,
2015
Zanamivir Powderinhalation Intranasal(L) Intranasal(L)/Intraperitoneal Herlocheretal.,2003;Hurtetal.,2010;Smee
etal.,2012a,b
Laninamivir Powderinhalation Intranasal(L)/Intratracheal(P) Intranasal(L) Kuboetal.,2010;Panozzoetal.,2015
Peramivir Intravenous Intravenous Intravenous/Intramuscular/Oral Yunetal.,2008;Bantiaetal.,2011;Kitanoetal.,
2011;Smeeetal.,2012a
T-705 Oral NR Oral Kisoetal.,2010
DAS181a Powderinhalation NR Intranasal(L) Marjukietal.,2014
aInvestigationaldrug;NR,Notreported;L,Liquid;P,Powder.
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OhandHurt AntiviralEffectivenessUsingtheFerret
dose of drug being administered. In humans, the optimal dose of a consensus dose in an animal to allow a better assessment
is determined by pharmacokinetic/pharmacodynamic (PK/PD) andcomparisonoftheeffectivenessofthedrugsacrossdifferent
analysis. The PK/PDof influenza antiviralsis often determined studies.
during pre-clinical testing in animals by pharmaceutical
companies (Urso et al., 2002), but data from these studies is
PARAMETERS TO ASSESS THE
often not in the public domain. For example, the majority of
studies have used 5 mg/kg oseltamivir phosphate in ferrets EFFECTIVENESS OF ANTIVIRAL
as being ‘equivalent’ to the 75 mg dose in humans, although TREATMENT
limited publicly available PK/PD data is available to support
this (Li et al., 1998; Mendel et al., 1998; Reddy et al., 2015). Pathology
At the time of writing this review, we have found only a small
One advantage of using the ferret is the ability to track viral
number of PK studies of the influenza antivirals in animals
sheddingkineticsfromboththeupperand/orlowerrespiratory
such as mice, rats, and ferrets (Table 3). As the generation
tract via nasal washing or lower bronchoalveolar lavage (BAL),
and analysis of PK/PD data can involve a large number of
although the latter is rarely used (Lee et al.,2014). In addition,
animals and requires specialized modeling expertise, it is not
ferrets can be monitored for clinical symptoms associated with
alwaysaviableoptionforresearchlaboratoriestocompletesuch
human influenza infections including weight loss, rise in body
studies. Therefore, if PK/PD data of approved drugs in ferrets
temperature, sneezing, nasal discharge, and lethargy (Table 4).
or other animals was made publicly available in open access
Whilst weight, temperature and symptoms such as sneezing
databases, then this would greatly assist and encourage the use
and nasal discharge can be easily assessed, lethargy is more
challengingtoquantitateaccurately.Conventionally,theactivity
of ferrets has been determined by visually assessing movement
TABLE3|Pharmacokineticsreportsofdifferentinfluenzaantiviralsin of the animal and assigning an arbitrary score (Reuman et al.,
mice,rats,andferrets. 1989;Govorkovaetal.,2011;Kimetal.,2013;Starketal.,2013;
Marriottetal.,2014;Ohetal.,2014).However,wehaverecently
Drugs Mice/Rats Ferrets
reportedontheuseofvideo-trackingmethodology,whichallows
Amantadine NR NR theactivitylevelofferretstobequantitatedbycomputersoftware
Rimantadine Hoffmanetal.,1988 NR analysis (Oh etal., 2015). Not only did video-tracking improve
Oseltamivir Lietal.,1998 Lietal.,1998;Reddyetal.,2015 thesensitivityofdetectingactivitychangespost-infection,italso
Zanamivir Lietal.,1998 NR enabled the assessment process to be simplified and less prone
Laninamivir Koyamaetal.,2009,2010 NR to bias. To date, many studies including ours have shown that
Peramivir Kodamaetal.,2014 Kitanoetal.,2011 treatment with an antiviral, such as oseltamivir, can improve
T-705 NR NR the activity level of ferrets infected with influenza (Govorkova
DAS181 NR NR et al., 2011; Marriott et al., 2014; Oh et al., 2014, 2015). This
NR,Notreported. improved methodology allows a more accurate assessment and
TABLE4|Differenttypesofclinicalandsymptomaticparametersmeasuredininfluenzastudiesinferrets.
Parameters Reference
Symptomatic Sneezing Huangetal.,2011,2012;Robertsetal.,2012;Kimetal.,2013;Paquetteetal.,2014
Weightloss Govorkovaetal.,2011;Huangetal.,2011,2012;Kimetal.,2013;vanderVriesetal.,2013;Marriott
etal.,2014;Ohetal.,2014,2015;Paquetteetal.,2014;Panozzoetal.,2015
Bodytemperature Govorkovaetal.,2011;Huangetal.,2011;Robertsetal.,2012;Kimetal.,2013;vanderVriesetal.,
2013;Marriottetal.,2014;Ohetal.,2014,2015;Panozzoetal.,2015
Activity(manualscoring) Govorkovaetal.,2011;Huangetal.,2011;Kimetal.,2013;Marriottetal.,2014;Ohetal.,2014
Activity(video-tracking) Ohetal.,2015
Virological Viralsheddingkineticsa,b,c Govorkovaetal.,2011;Huangetal.,2012;Kimetal.,2013;vanderVriesetal.,2013;Marriottetal.,
2014;Ohetal.,2014,2015;Paquetteetal.,2014;Panozzoetal.,2015
Histopathological Lunghistology Cameronetal.,2008;Roweetal.,2010;Huangetal.,2011,2012;Kimetal.,2013
Inflammation Totalviablecellcounta Govorkovaetal.,2011;Marriottetal.,2014;Ohetal.,2014,2015;Panozzoetal.,2015
Totalproteinconcentrationa Govorkovaetal.,2011;Ohetal.,2014,2015;Panozzoetal.,2015
Immunological Differentialcellcount(innateimmunecells)c Kimetal.,2013
Influenza-specificantibodies vanderVriesetal.,2013;Ohetal.,2014,2015;Panozzoetal.,2015
Hematologychemistry Starketal.,2013
RNAexpression(RT-PCR) Kimetal.,2013;Carolanetal.,2014,2015;Paquetteetal.,2014
RNAexpression(Microarray) Cameronetal.,2008;Roweetal.,2010
aNasalwash;bAssaybyTCID50,PFUorRT-PCR;cBronchoalveolarlavage.
FrontiersinMicrobiology|www.frontiersin.org 7 February2016|Volume7|Article80
OhandHurt AntiviralEffectivenessUsingtheFerret
extrapolation of the associated effect of antivirals on activity or CD8+ T cells in mice, they had normal recall responses and
‘wellness’. conferred protection against subsequent infection (Bird et al.,
2015).
Immunology
Themajordisadvantageofusingferretsasamodelforstudying FUTURE DIRECTIONS FOR THE USE OF
influenza (or influenza antiviral effectiveness) is the lack of
FERRETS TO ASSESS ANTIVIRAL
validated ferret-specific reagents, such as antibodies, to study
EFFECTIVENESS
the immunology of disease pathogenesis. However, because of
the significant benefitsof the modelmanylaboratories are now
To date, the ferret remains as one of the preferred models
attempting to validate cross-reactive antibodies and develop
for assessing influenza infection and as such has become an
novelreagentsforferretresearch(Rutiglianoetal.,2008;Martel
important modelforantiviraltesting.Althoughthe ferretposes
and Aasted, 2009; Music et al., 2014). One such collaborative
manyadvantagescomparedtootheranimalmodels,thisreview
effort is chaired by the United States Centre of Excellence for
has highlighted some of the challenges of using the ferret
InfluenzaResearchandSurveillancegroupatSt.JudeChildren’s
to assess antiviral effectiveness. Researchers conducting studies
Research Hospital in Memphis, USA. To date, several groups
in the ferret model of influenza infection need to consider
haveassessedferrethostimmuneresponsestoinfluenzainfection
progressing toward a more ‘natural’ infection methodology by
basedonRNAexpressionbyeitherquantitativereal-timePCRin
eithercontactornon-contacttransmission,oraerosolchallenge,
eithernasalwashes,blood orlung(Mainesetal.,2012;Carolan
using drug doses that are based on PK/PD analysis and
et al., 2014, 2015; Paquette et al., 2014; Vidana et al., 2014), or
to deliver the drugs via a relevant route (Figure 3). The
by using a canine microarray platform (Cameron et al., 2008;
development of more robust parameters to measure antiviral
Roweetal.,2010;Table4).Recently,thereportofanannotated
effectiveness in ferrets, such as computational analysis of
ferret transcriptome during influenza infection hasalso opened
behavior/activityandthedevelopmentofferret-specificreagents
up an avenue for interrogating the global host gene expression
to explore the immunological effects of antiviral treatment,
tofurtherunderstandhostimmunitytowardinfluenzainfection
will both greatly improve our understanding of antiviral
(Leon et al., 2013). In addition, simple immunological analysis
modes of action and the effect on viral pathogenesis. In our
such as measuring total cell count or protein concentration
opinion, the application of ferrets in antiviral studies should
in nasal wash samples can also be useful as a determinant of
not be limited to just understanding therapeutic effectiveness.
inflammation(Govorkovaetal.,2007,2011;Marriottetal.,2014;
Instead, the ferret model can be applied to address other
Oh et al., 2015; Panozzo et al., 2015). Other basic immune
questions such as the in vivo effectiveness of antivirals against
measurementsincludedifferentialstainingofvariousleukocytes
drug ‘resistant’ viruses, and the effectiveness of different
populations in the BALor blood, and alsoclinical chemistry of
antiviral treatment or prophylaxis strategies on preventing or
different proteins in blood post influenza infection (Kim et al.,
minimizing transmission. Therefore, we anticipate that with
2013;Starketal.,2013;Table4).
continuous development and refinement of the ferret model
Despiteagreatefforttostudytheinterplaybetweeninfluenza
for influenza antiviral testing, it has the potential to provide
infection and immunity in ferrets, the effect of antivirals on
more meaningful data tobetterinform humanuse of influenza
immunity in this modelis still poorly understood. To date,the
antivirals.
majorityoftheimmunity-relatedstudiesoninfluenzaantivirals
haveusedmice astheanimalmodel(Burgeretal.,2000;Wong
et al., 2011; Bird et al., 2015; Marois et al., 2015). Oseltamivir
AUTHOR CONTRIBUTIONS
was found to reduce innate cell populations in BAL, such
as neutrophils and macrophages, when given prophylactically,
Both authors made substantial, direct and intellectual
and reduced pro-inflammatory cytokines when given pre- and
contributiontothework,andapproveditforpublication.
post-infection (Wong et al., 2011). In contrast, the effect of
oseltamivir on the adaptive immune cell population, such as
+
cytotoxic CD8 T cells, is still unclear. While Burger et al. FUNDING
(2000) reported that oseltamivir had minimal impact on the
splenic population of T and B cells, and had no effect on DO is supported by a National Health and Medical Research
+
the cytolytic activity of CD8 T cells and natural killer cells Grant, Australia (APP 1055793). The Melbourne WHO
in A(H1N1) influenza-infected mice (Burger et al., 2000), a CollaboratingCentreforReferenceandResearchonInfluenzais
recent study byMaroisetal.(2015), found oseltamivir reduced supportedbytheAustralianGovernmentDepartmentofHealth.
+
circulatingandtissue-residenteffectorandmemoryCD8 Tcells
in A(H1N1) influenza-infected mice and significantly reduced
the protective effect in a subsequent infection episode (Marois ACKNOWLEDGMENT
et al., 2015). In addition, a study by Bird et al. (2015) showed
that although oseltamivir treatment reduced the number of WethankChantalBaasforcontributingferretviralloaddata.
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OhandHurt AntiviralEffectivenessUsingtheFerret
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FrontiersinMicrobiology|www.frontiersin.org 10 February2016|Volume7|Article80