In ovo and in vitro susceptibility of American alligators (Alligator mississippiensis) to avian...

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IN OVO AND IN VITRO SUSCEPTIBILITY OF AMERICANALLIGATORS (ALLIGATOR MISSISSIPPIENSIS) TO AVIANINFLUENZA VIRUS INFECTIONAuthor(s): Bradley L. Temple, John W. Finger, Jr., Cheryl A. Jones, Jon D. Gabbard, TomislavJelesijevic, Elizabeth W. Uhl, Robert J. Hogan, Travis C. Glenn, and S. Mark TompkinsSource: Journal of Wildlife Diseases, 51(1):187-198.Published By: Wildlife Disease AssociationURL: http://www.bioone.org/doi/full/10.7589/2013-12-321

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IN OVO AND IN VITRO SUSCEPTIBILITY OF AMERICAN ALLIGATORS

(ALLIGATOR MISSISSIPPIENSIS) TO AVIAN INFLUENZA

VIRUS INFECTION

Bradley L. Temple,1,6 John W. Finger Jr.,1,6 Cheryl A. Jones,2 Jon D. Gabbard,2

Tomislav Jelesijevic,3 Elizabeth W. Uhl,3 Robert J. Hogan,2,4 Travis C. Glenn,1

and S. Mark Tompkins2,5

1 Department of Environmental Health Science, University of Georgia, 150 Green St., Athens, Georgia 30602, USA2 Department of Infectious Diseases, University of Georgia, 111 Carlton St., Athens, Georgia 30602, USA3 Department of Pathology, University of Georgia, 501 D. W. Brooks Dr., Athens, Georgia 30602, USA4 Department of Anatomy and Radiology, University of Georgia, 111 Carlton St., Athens, Georgia 30602, USA5 Corresponding author (email: [email protected])6 These authors contributed equally to this manuscript.

ABSTRACT: Avian influenza has emerged as one of the most ubiquitous viruses within ourbiosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses;however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understandingwhich and how many species can be infected. Moreover, novel influenza virus sequences wererecently isolated from New World bats. Crocodilians have a slow rate of molecular evolution andare the sister group to birds; thus they are a logical reptilian group to explore susceptibility toinfluenza virus infection and they provide a link between birds and mammals. A primary Americanalligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenicavian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggssupported virus replication, as evidenced by the influenza virus M gene and infectious virusdetected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cellswere also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining;however, the requirement for trypsin to support replication in cell culture limited replication. Toassess influenza virus replication in culture, primary alligator cells were inoculated with H1N1human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both virusesreplicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. Thisresearch demonstrates the ability of wild-type influenza viruses to infect and replicate within twocrocodilian substrates and suggests the need for further research to assess crocodilians as a speciespotentially susceptible to influenza virus infection.

Key words: Alligator mississippiensis, avian influenza virus, crocodilian, eggs, embryonicfibroblasts, reptile, susceptibility.

INTRODUCTION

The emergence of zoonotic influenza Avirus, including highly pathogenic avianinfluenza (HPAI) viruses H5N1 and pan-demic H1N1 influenza viruses, hassparked worldwide interest in identifyingand understanding which and how manyspecies can be infected and serve asreservoir or vector species for influenzaA virus. While influenza A virus has beenwell documented in mammalian and avianspecies, new viruses and host species arestill being identified, most recently in bats(Tong et al. 2012; Tong et al. 2013).Recent research suggests that insects(Barbazan et al. 2008), reptiles (Huchzer-

meyer 2002; Davis and Spackman 2008),and amphibians are also susceptible toinfluenza infection (Huchzermeyer 2002;Mancini et al. 2004; Barbazan et al. 2008;Davis and Spackman 2008). Four speciesof crocodilian, the Chinese alligator (Alliga-tor sinensis), smooth-fronted caiman (Paleo-suchus trigonatus), broad-snouted caiman(Caiman latirostris), and Nile crocodile(Crocodylus niloticus), have evidence ofinfluenza A susceptibility, with portions ofthe virus genome identified by PCR analysisof blood and serum of captive crocodilians(Davis and Spackman 2008). In addition,influenza C was identified by electronmicroscopy in the feces of farmed Nilecrocodiles (Huchzermeyer 2003). To our

DOI: 10.7589/2013-12-321 Journal of Wildlife Diseases, 51(1), 2015, pp. 187–198# Wildlife Disease Association 2015

187

knowledge, only samples from captivecrocodilians have been analyzed; thus,data are deficient for wild crocodilians.Susceptibility to infection through directinoculation of crocodilian tissues, cells, orlive animals has not been investigated.

Avian species, mainly Anseriformes(e.g., ducks) and Charadriiformes (e.g.,gulls), are the natural reservoirs of influ-enza A viruses (Hubalek 2004; Krauss etal. 2007). Evolutionarily, crocodilians aremost closely related to birds (Zardoya andMeyer 2001; Crawford et al. 2012).Crocodilians and birds share several phys-iologic features such as egg structure andembryonic development (Deeming andFerguson 1991), similar antibody isotypes(Warr et al. 1995), and homologouspancreatic polypeptides (Lance et al.1984; Deeming and Ferguson 1991; Warret al. 1995). Moreover, birds and croco-dilians demonstrate similar susceptibilityto some pathogens including West Nilevirus, caiman pox virus, crocodile poxvirus, adenoviruses, Newcastle diseasevirus, eastern equine encephalitis virus,and coronaviruses (Ritchie 1995; Huch-zermeyer 2003; Klenk et al. 2004; Davisand Spackman 2008). Therefore, it isreasonable to hypothesize that crocodil-ians may be susceptible to other virusesendemic to birds, including influenzaviruses, and could potentially serve as areservoir or mixing vessel for these viruses.

Extant crocodilians are traditionallydivided into three families includingAlligatoridae, Crocodylidae, and Gaviali-dae, with the Alligatoridae includingalligators and caimans (Janke et al. 2005;St. John et al. 2012). American alligators(Alligator mississippiensis) live in proxim-ity to and opportunistically feed on variousavian species (Elsey et al. 2004). Thus,alligators may routinely be exposed toavian influenza virus (AIV) through theingestion of infected tissues and inhalationof infectious particles during feeding(Reperant et al. 2008). Alligators are alsoexposed to bird excrement. As the fecal-oral route is considered the primary route

of influenza virus transmission in aquaticbirds (Wright et al. 1992), this mayprovide an additional route of infectionfor alligators.

The shared biologic and ecologic fea-tures of alligators and aquatic birds makealligators an important animal to investigateas a potential mixing vessel or reservoir forAIV. In addition, with increasing wild andfarm populations, along with habitat en-croachment, human-alligator interactionsare increasing. We used primary alligatorcells and alligator embryos to assess thesusceptibly of American alligators to influ-enza A virus infection.

MATERIALS AND METHODS

Viruses

The low pathogenic avian influenza (LPAI)viruses used in this study were: A/chicken/Texas/167280-4/02 (H5N3), A/blue-wingedteal/Louisiana/69B/87 (H4N8), A/mallard/Minnesota/Sg-00692/08 (H2N3), and A/blue-winged teal/Louisiana/Sg-00224/07 (H3N8).Notably, the H3N8 strain was isolated fromthe same region where the alligator eggs forthis study were collected. The LPAI viruseswere provided by David Stallknecht (Univer-sity of Georgia, Athens, Georgia, USA). Thehuman influenza A virus (A/WSN/1933[H1N1]) is a mouse-adapted virus that repli-cated in cell culture without trypsin whenserum is present (Goto et al. 2001). The HPAIvirus, A/Viet Nam/1203/2004 (H5N1), wasprovided by Richard Webby (Saint JudeChildren’s Research Hospital, Memphis, Ten-nessee, USA). The viruses were propagated at37 C in 9–11-d-old embryonated chicken eggs.Virus titers were assayed in Madin Darbycanine kidney (MDCK) cells by 50% tissueculture infectious dose (TCID50) assay (Sobo-leski et al. 2011; Mooney et al. 2013) andranged from 104.50 to 106.24 TCID50/mL. Allexperiments using live HPAI viruses wereapproved by the institutional biosafety pro-gram at the University of Georgia and wereconducted in biosafety level 3–enhancedcontainment following guidelines for use ofSelect Agents approved by the US Centers forDisease Control and Prevention.

Tissue culture

Primary American alligator embryonic fi-broblasts were established from a 41–51-d-old

188 JOURNAL OF WILDLIFE DISEASES, VOL. 51, NO. 1, JANUARY 2015

embryo. The embryo was chilled overnight at4 C, dissected from the egg, washed withalpha-MEM (Gibco, Carlsbad, California,USA) and 13 antibiotics (100 IU/mL penicil-lin G, 100 mg/mL streptomycin, 0.25 mg/mLamphotericin B), cut into 131-mm segments,and washed three times in MEM 13 antibi-otic/antimycotic mixture. Segments were di-gested in a 1:10 dilution of collagenase B(Roche, Indianapolis, Indiana, USA) in alpha-MEM cell culture media containing antibiotics(penicillin/streptomycin, amphotericin B; al-pha-MEM/AB media) for 3 h. Suspensionswere filtered through a 70-mm cell strainer(BD Falcon, San Jose, California, USA),washed with alpha-MEM/AB media, andcentrifuged at 1,500 3 G for 15 min. Cells,which were presumed to be fibroblasts basedon morphologic characteristics, were culturedin the alligator cell culture media (ACCM) forall experiments unless otherwise stated(ACCM: 175 mM MEM [Gibco], Primocin(100 mg/mL [Invivogen] San Diego, California,USA), 2 mM L-glutamine (HyClone, SouthLogan, Utah, USA), 13 penicillin/streptomy-cin, amphotericin B (CellGro, Manassas,Virginia, USA), 10% fetal bovine serum(FBS), 25 mM HEPES (Gibco), and 13sodium bicarbonate (7.5 mg/mL; Gibco) atpH 7.5 (Cuchens and Clem 1979). Cells wereincubated at 30 C and 6% CO2.

Egg inoculation

Alligator eggs were collected from Rock-efeller Wildlife Refuge (Cameron and Vermil-ion Parishes, Louisiana, USA, 29u739N,92u829W). The University of Georgia Institu-tional Animal Care and Use Committeeapproved all protocols involving animals. Eggswere incubated in a mixture of moist vermic-ulite and peat moss at 28–30 C and 90%humidity.

Viability was determined by candling. Viableeggs were segregated into four groups of 10infected eggs and one group of 12 controleggs. Of the infected groups, five eggs wereincubated at 33 C and another five at 36 C(33 C is the optimum temperature for normalalligator embryonic development; 36 C is theclosest optimal temperature for virus replica-tion without inducing embryonic lethality;Smith 1975). The control eggs were dividedequally into temperature controls and vehiclecontrols (three untreated and three givenvehicle and incubated at 33 C; three untreatedand three given vehicle and incubated at 36 C).The injection site was disinfected with 70%ethanol prior to inoculation. Virus diluted(1:100) in phosphate-buffered saline (PBS)

and antibiotics (100 IU penicillin G, 100 mg/mL streptomycin, 0.25 mg amphotericin B/mL)were injected blindly (the allantoic fluid cavitywas targeted but not confirmed) using a sterile24-mm 18-ga needle. The injection site wassealed with Elmer’s glue and eggs wereincubated for 5 d at indicated temperatures.Prior to extraction, eggs were chilled overnightat 4 C. Embryos were placed into 50 mLsterile conical tubes and filled with virustransport media (VTM; MEM, 1003 antibiot-ic/antimycotic [10,000 IU/mL penicillin,10,000 mg/mL streptomycin, 25 mg/mL am-photericin B], 50 mg/mL gentamicin, 50 mg/mL kanamycin, pH 7.4). The allantoic fluidsamples and whole embryos were stored at280 C. The control embryos were cut toreveal viscera and stored at room temperaturein 10% formalin.

Virus titration

Virus titration of allantoic fluid and thetissues from embryonated alligator eggs wereassayed by TCID50 assay using a cell-basedenzyme-linked immunosorbent assay (ELISA).Embryos were thawed at 4 C for 8 hr; tissueswere extracted from embryos and homoge-nized in VTM using QiagenH Tissue Lyser(Germantown, MD, USA).

For the TCID50 assay, MDCK cells platedin 96-well micro-titer plates were washed andreplaced with MDCK infection medium(MEM, 2 mM L-glutamine, 13 antibiotic/antimycotic [100 IU/mL penicillin, 100 mg/mLstreptomycin, 25 mg/mL amphotericin B],50 mg/mL gentamicin, and 1 mg/ml tosylphenylalanyl chloromethyl ketone [TPCK]trypsin [Worthington, Lakewood, New Jersey,USA]). Tissue homogenates were added intriplicate followed by 10-fold serial dilutions inthe remaining rows. Negative (uninfectedculture medium) and positive controls consti-tuted the last row, with the positive controlsinfected with 500–750 TCID50 of virus. Plateswere covered and incubated for 36 h or 72 h(37 C, 5% CO2) and fixed with methanol/acetone (80:20).

Plates were blocked overnight (5% nonfatdry milk, 0.5% bovine serum albumin [BSA],and 13 KPL wash buffer; KPL, Gaithersburg,Maryland, USA) at 4 C, washed three times,and incubated 1 h with mouse anti-influenzanucleoprotein (NP) immunoglobulin G (IgG;H16-L10; provided by Jon Yewdell, NIAID,Bethesda, Maryland, USA). After washing,anti-NP binding was detected using goatanti-mouse IgG horseradish peroxidase(HRP) conjugate (1:10,000 dilution in KPLwash buffer) and incubated in the dark at

TEMPLE ET AL.—SUSCEPTIBILITY OF AMERICAN ALLIGATORS TO AIV INFECTION 189

room temperature for 1 h, followed by washingand detection using 1-Step Ultra TMB(3,39,5,59-tetramentylbenzidine) ELISA sub-strate solution (Pierce, Rockford, Illinois,USA) following the manufacturer’s directions.Sulfuric acid was added to stop the TMBreaction and plates were read at 450 nm usinga BioTek Powerwave plate reader (Bio-TEK,Winooski, Vermont, USA). For some samples,the virus titer was also estimated by measuringhemagglutinin (HA) of MDCK culture super-natants with 0.5% turkey red blood cells(Soboleski et al. 2011; Mooney et al. 2013).Briefly, 0.05 mL of supernatant from each wellof the TCID50 plate was added to 0.05 mL of0.5% red blood cells (diluted in PBS) andassayed for agglutination within 1 h. The 50%endpoint was calculated via the Reed andMuench method (Reed and Meunch 1938).

Immunofluorescence staining

Alligator fibroblast cells were trypsinizedusing 0.05% Trypsin-EDTA and then plated at7.53105 cells/mL in ACCM in 12-well plates.Once cells reached 80–90% confluence, themedium was removed and virus diluted(multiplicity of infection [MOI]+0.05) in virusinfection media (ACCM without FBS andHEPES plus 1 mg/mL TPCK-trypsin wasadded to each well). Inoculated plates wereincubated for 3 h at 30 C and 6% CO2.Subsequently, virus infection media was re-moved, plates were washed with sterile PBS,and alligator cell growth medium (withouttrypsin) was added to each well. Plates wereincubated at 33 C under 6% CO2 for 24 h and72 h or at 37 C under 6% CO2 for 24 h.Following incubation, plates were washed inPBS and fixed for 20 min with methanol/acetone (80:20). Plates were blocked (5%FBS, 0.1% TweenH20, and 13 KPL washbuffer) overnight at 4 C, washed three times,and incubated for 3 h at room temperaturewith mouse anti-influenza NP IgG (H16-L10).Plates were washed and goat anti-mouse IgGFITC-conjugated in 13 PBS was added toeach well. Plates were incubated at roomtemperature for 1 h in the dark, washed, and1 mg/mL of 49,6-diamidino-2-phenylindole wasadded to each well to visualize cell nuclei.Plates were incubated for 20 min at roomtemperature in the dark, washed, and exam-ined using a Zeiss Axiovert 40 CFL fluorescentlight microscope (Carl Zeiss Microscopy,Thornwood, New York, USA).

Assay for in vitro replication

Alligator fibroblasts were plated at 104 cells/well in ACCM in a 24-well plate. At 60%

confluence, ACCM was removed and cellswere washed three times with 13 MEM.Fibroblasts were infected with 100 plaque-forming units (PFU) of A/WSN/33 (H1N1) orA/Viet Nam/1203/04 (H5N1) diluted inACCM without HEPES for 3 h at 37 C, 5%CO2. Following incubation, cells were washedthree times with 13 MEM, and 1 mL ofACCM without HEPES was added to eachwell and plates were incubated at 30 C, 6%CO2. At 24, 48, and 72 h, supernatants werecollected, clarified by centrifugation, BSAadded to 0.2%, aliquoted, and stored at280 C. Infectious virus titers in supernatantswas determined by TCID50 assay, as de-scribed, except that determination of influenzavirus-positive wells was determined by HA.

Immunohistochemistry

Formalin-fixed alligator organs (brain, tra-chea, lung, heart, liver, intestine, stomach,kidney, spleen, and pancreas) were dissectedfrom embryos, embedded in paraffin, sec-tioned, and stained with H&E for histologicanalysis. Other sections were rehydrated usingstandard procedures and stained using mouseanti-influenza A NP IgG, biotin conjugated(Bioss Inc., Woburn, Massachusetts, USA),followed by a streptavidin-HRP labeled sec-ondary antibody. Lastly, 3,39-diaminobenzi-dine was added to produce a brown precipitatein antigen-positive tissues. Sections werereviewed and scored by a board certifiedpathologist (E.W.U.).

Real-time reverse-transcriptase (RT)-PCR

Total RNA was extracted from the liver,kidney, and a subset of allantoic fluid samplesusing a Qiagen RNeasy Mini Kit, according tothe manufacturer’s protocol, except that a 250-mL aliquot of allantoic fluid was used in theextraction process (Spackman et al. 2002).

The cDNA synthesis and subsequent real-time RT-PCR were performed using a QiagenOneStep RT-PCR Kit on a StratageneMX3000p or 3005p thermocycler (AgilentTechnologies, Santa Clara, CA, USA) follow-ing the protocol of Spackman (2002). This is awell-established TaqManH (Life Technologies,Carlsbad, CA, USA) protocol used as aninfluenza A virus diagnostic assay. The primerprobe sequences are: M+25, AGA TGA GTCTTC TAA CCG AGG TCG; M–124, TGCAAA AAC ATC TTC AAG TCT CTG; andM+64, FAM-TCA GGC CCC CTC AAA GCCGA-TAMRA (Spackman et al. 2002). Eachsample was run in triplicate with influenza A/WSN/33 as a positive control.


Statistics

All statistical analyses were performed usingGraph Pad Prism software (GraphPad Software,Inc., La Jolla, California, USA). A Mann-Whitney test or analysis of variance, followedby Bonferroni’s multiple comparison test, wereused to determine statistical significance. Thelevel of significance for all data was set at ,0.05.

RESULTS

In vitro infection of primary embryonic fibroblasts

Alligator primary embryonic fibroblastswere generated as described in theMethods and infected to determine sus-ceptibility to AIV infection. Following inoc-ulation (MOI50.05) with A/blue-wingedteal/Louisiana/Sg-00224/07 (H3N8), prima-ry embryonic alligator fibroblasts werepositive for virus NP (Fig. 1). The NPantigen was present within the cytoplasmbut was more strongly co-localized in thenucleus. Cells infected with AIV werepositive for NP antigen after incubation at33 C or 37 C at 24 h postinfection. Infectedalligator fibroblasts were also positive for NPat 72 h postinfection at 33 C incubation.Infection with A/blue-winged teal/Louisi-ana/69B/87 (H4N8), A/chicken/Texas/167280-4/02 (H5N3), or A/mallard/Minne-sota/Sg-00692/08 (H2N3) showed similarresults (data not shown).

Infection of embryonated alligator eggs with AIV

Embryonated alligator eggs were inoc-ulated with one of the four LPAI (H5N3,H4N8, H3N8, or H2N3) strains to deter-mine susceptibility to virus replication inovo. Allantoic fluid and embryos wereextracted 5 d postinoculation to assess thesusceptibility to infection.

Infectious virus titers postinoculationexceeded input virus for all four strains, asindicated by virus titer levels in allantoicfluid of embryonated alligator eggs (Fig. 2).The H5N3, H4N8, and H3N8 strains ofinfluenza virus incubated at 33 C had meanvirus titers significantly greater than inputvirus (P,0.001) with values of 104.79

(6100.77), 103.84 (6100.50), and 103.59 (6100.55)TCID50/mL, respectively (mean [6SEM]),

whereas at 36 C these three strains hadmean virus titers of 104.84 (6100.65), 104.35

(6100.15), and 104.39 (6100.57) TCID50/mL,respectively. This suggests that tempera-tures chosen for replication had no signif-icant impact on virus replication whencultured in alligator eggs. In contrast, theH2N3 virus demonstrated improved repli-cation at the higher temperature, with the33 C group having a mean virus titer of103.49 (6100.67) TCID50/mL compared to106.04 (6100.67) TCID50/mL at 36 C(P50.032). To validate these results, asubset of allantoic fluid samples wasassayed in a standard TCID50 assay usingHA as a measure of virus replication(Tompkins et al. 2007; Soboleski et al.2011). The virus titer, as measured by HAendpoint, corresponded almost directlywith the titers measured by cell-basedELISA (data not shown).

Virus genomic RNA was measured in asubset of allantoic fluid samples (n55) fromthe 36 C alligator eggs. The RNA waspurified from allantoic fluid and M genegenomic RNA levels were measured usingreal-time RT-PCR. The mean cycle thresh-old (Ct) values from allantoic fluid samples(Fig. 3) were below the negative thresholdlevel of 34.79 (60.17). H5N3-infected sam-ples assayed had a mean Ct value of 13.97(60.15), while the H3N8- and H4N8-infected samples had mean Ct values of15.38 (60.13) and 15.25 (60.06), respective-ly. The H2N3-infected samples had thelowest mean Ct value, 13.23 (60.27), corre-sponding to the highest titer measured in theTCID50 assay (Fig. 2). A/WSN/33 (H1N1),used as a positive control, had a mean Ct

value of 15.67 (60.37). Taken together, theinfectious virus assay and M gene PCR dataindicate influenza virus replication occurredwithin the alligator eggs.

AIV infection in embryonic tissues

After confirmation of infection and virusreplication of all four AIV strains in theallantoic fluid of embryonated alligatoreggs, localization of replication was deter-mined. Organs were extracted from em-


bryos for H&E staining, IHC, virusculture, and RNA isolation.

All mock-infected embryos were nega-tive for congestion and necrosis in alltissues and had no indications of antigenstaining. However, one vehicle control at36 C had fluid within the lungs, resulting innonspecific staining, and so was excludedfrom further analysis (data not shown). Incontrast, the alligator eggs infected witheach of the four virus strains had NPstaining predominantly in liver and kidneysand, in accordance with previous results,

had higher levels of staining at 36 C.Embryos inoculated with each respectivestrain were positive for NP antigen stainingin liver, kidney, or both. Likewise, positivestaining was present at 33 C and 36 C.Embryos illustrated necrosis, congestion,and lesions in kidney and liver and in othertissues with all four virus strains (Table 1).

The IHC analysis indicated that theliver and kidney might serve as predom-inant sites for virus replication. However,attempts to measure tissue virus titersfrom embryos by TCID50 were unsuccess-

FIGURE 1. Immunofluorescent staining of nucleoprotein (NP) antigen in primary alligator (Alligatormississippiensis) fibroblasts infected with avian influenza virus H3N8. Positive staining of NP at a multiplicityof infection of 0.05, 24 h postinfection at 33 C, at 403 (A) and 103 (B) magnification. Co-localization of 49, 6-diamidino-2-pheylindole and NP 24 h postinfection at 33 C at 403 (C) and 103 (D) magnification.


ful. To confirm the results obtained fromthe IHC, real-time RT-PCR was utilized.Total RNA was extracted from bothkidney and liver from the remainingembryos in each group and M gene RNAlevels were compared with controls. Themean control Ct values from the uninfect-ed embryonic tissues were used to calcu-late the negative threshold value (36.40).The control values for the embryosincubated at 33 C were 40.00 (61.03)for kidney (n59) and 42.19 (61.14) forliver (n59; mean [6SD]). Embryos incu-bated at 36 C had similar values of 40.65(61.03) for kidney (n512) and 41.80(60.75) for liver (n512). The mean Ct

value of liver and kidney samples fromembryos infected with each of the fourstrains at two respective temperatureswere below the negative threshold, indi-cating the presence of M gene genomicRNA and virus replication in inoculatedembryos (Fig. 4). Temperature of incuba-tion did not affect virus replication forH4N8, H3N8, or H5N3 AIV. In contrast,consistent with previous results, H2N3-inoculated embryos incubated at 36 C hadincreased M gene RNA compared withembryos incubated at 33 C (Fig. 4c).

Replication of influenza virus in alligatorfibroblast cells

As the LPAI viruses were able toreplicate in alligator embryos, we nextdetermined the ability of alligator primaryembryonic fibroblasts to support thecomplete influenza virus lifecycle. Influ-enza virus generally requires addition ofexogenous trypsin or another protease toactivate the HA of progeny virions,enabling multiple replication cycles (Shawand Palese 2013; Wright et al. 2013).These proteases can be damaging to cellsin culture, limiting the ability to assessinfluenza virus replication in many cellsubstrates. However, A/WSN/33 (H1N1)influenza can replicate efficiently withoutthe addition of trypsin through sequestra-tion of plasminogen in exogenously addedserum (Goto et al. 2001). We infectedalligator fibroblasts with 100 PFU of A/WSN/33 in media containing serum,cultured the infected cells at 30 C, andcollected supernatants over time. The

FIGURE 2. Mean virus titer in allantoic fluid fromembryonated alligator (Alligator mississippiensis)eggs. Embryonated eggs (n55/group) were inoculat-ed with the indicated virus and incubated for 5 d atthe indicated temperatures (33 C or 36 C). Allantoicfluid was collected and assayed for infectious virus by50% tissue culture infectious dose (TCID50) assay(*statistically significant, P,0.05).

FIGURE 3. Real-time reverse-transcription-PCRof M gene levels from alligator (Alligator mississip-piensis) allantoic fluid infected with low pathogenicavian influenza virus. Embryonated eggs wereinoculated with influenza virus and incubated for5 d at 36 C. Allantoic fluid was collected and assayedfor virus genome. Allantoic fluid was collected fromnaı̈ve eggs as a negative control. Allantoic fluid fromA/WSN/33-infected embryonated chicken eggs wasused as a positive control. All infected samples werebelow the negative control cycle threshold (Ct) of34.79. Control (n59); H5N3, H4N8, and, H3N8(n56); H2N3 (n53); and WSN (n59).


supernatants were assayed for infectiousvirus by TCID50 assay. At 24 h postino-culation there was limited, albeit measur-able, infectious virus detected (Fig. 5a).The lower culture temperature was likelyprolonging the eclipse phase of virusreplication. However, at 48 h and 72 hpostinoculation, there was significantlymore infectious virus (P50.003), demon-strating replication of A/WSN/33 in alliga-tor cells.

A hallmark of HPAI viruses is the abilityto replicate in culture in the absence oftrypsin. The HA proteins of HPAI viruseshave polybasic cleavage sites (in contrastto the monobasic cleavage sites found inmost influenza viruses) that enable cleav-age and activation of the HA protein byubiquitous, furin-like proteases found inthe Golgi complex of eukaryotic cells(Shaw and Palese 2013; Wright et al.2013). To assess the ability of an avianinfluenza virus to replicate in alligators, weinfected fibroblasts with 100 PFU of A/Viet Nam/1203/2004 (H5N1) and col-lected and assayed supernatants as before.Similar to the H1N1 virus, there waslimited virus at 24 h but significantincreases in infectious virus at 48 h and72 h postinoculation (P,0.001), confirm-

ing that alligator cells are susceptible toAIV infection and can support the com-plete infectious lifecycle (Fig. 5b).

DISCUSSION

We demonstrated in ovo and in vitrosusceptibility of American alligators toLPAI virus infection, with infection andreplication occurring at temperaturesmuch lower than wild-type AIVs wouldnormally replicate (Massin et al. 2001,2010; Scull et al. 2009). Measurement ofvirus load in allantoic fluid and culturesupernatants indicated robust replicationand production of virus progeny afterinfection with human influenza, LPAI,and HPAI viruses. While we demonstrat-ed in ovo and in vitro susceptibility toAIV in alligators, the ability of AIV toreplicate in live crocodilians remainsunknown.

Primary alligator fibroblast cells werepositive for NP after infection with eachstrain, demonstrating infection with mul-tiple subtypes and strains. The primaryalligator fibroblasts also supported pro-duction of infectious progeny viruses at30 C and 37 C (data not shown). While thetrypsin requirement that is common for

TABLE 1. Histopathology from embryonic tissues. Summary of histology and immunohistochemistry datafrom embryos extracted from inoculated alligator (Alligator mississippiensis) eggs. Numbers in parenthesesare number positive/number tested. Dashes (–) indicate no pathology or antigen staining found.

Virus Head Neck Chesta Abdomena NP antigena

Controlb – – – – –H3N8 33 C – – – – Lu, L, K (1/2)H3N8 36 C – – Lu congestion (1/2)

Lu necrosis (1/2)– L, K (2/2)

H4N8 33 C – – Lu congestion (2/2) K congestion (1/2) Lu (1/2); L, K (2/2)H4N8 36 C Necrosis (2/2) Necrosis (2/2)

Bacteria (1/2)Necrosis (2/2) Necrosis (2/2) Lu (1/2)

L, K (2/2)H5N3 33 C – – – – L, K, I (1/2)H5N3 36 C Necrosis (1/2)

Bacteriac (1/2)Necrosis (1/2) Necrosis (1/2) Necrosis (1/2) L, K (1/2)

H2N3 33 C – – Lu congestion (1/2) – L (1/2)H2N3 36 C – – – – L, K (2/2)

a NP 5 nucleoprotein; L 5 liver; Lu 5 lung; K 5 kidney;b Control embryos n54. Infected embryos n54 per virus strain. One control embryo in the vehicle control 36 C group

inhaled fluid, which resulted in nonspecific staining (false positive).c ‘‘Bacteria’’ indicates bacterial contamination.


growth of most influenza viruses in cellculture limited the viruses which could beassayed for replication, both human andavian influenza viruses replicated in thealligator cells, suggesting sialic acid moie-ties were present for HA binding.

All four AIVs replicated within theembryonated alligator eggs, with all fourstrains infecting liver and kidneys. Allvirus-inoculated eggs produced high virus

titers in the allantoic fluid. Virus titerswere several logs greater than input titersfor all four strains and at both tempera-tures, with the highest production at 36 C.Overall, these temperatures coincide with-in an optimal temperature range foralligator metabolic activity (Gans 1969).However, reptilian immune function isdependent upon temperature, with ex-treme temperatures perturbing function;

FIGURE 4. Reverse-transcription-PCR of M gene in infected embryonic tissues. Real-time RT-PCRresults of influenza A genomic RNA specific for M gene from kidney and liver samples of infected anduninfected alligator (Alligator mississippiensis) embryos. Embryonated eggs were inoculated with influenzavirus and incubated for 5 d at 33 C or 36 C. Eggs were necropsied and tissues collected and processed forRNA as described in the materials and methods. Tissues were collected from naı̈ve eggs as a negative control.All samples with a cycle threshold (Ct) value .36.40 were classified as negative for M gene. All samples with aCt ,36.40 were classified as positive for influenza A genomic RNA. A + indicates mean Ct value for allsamples (n59, except for 36 C control kidney and control liver, where n512). White box indicates anincubation temperature of 33 C; checkered box indicates incubation temperature of 36 C. (A) H4N8-infectedtissues. (B) H3N8-infected tissues. (C) H2N3-infected tissues. (D) H5N3-infected tissues.


thus, suppressed immune function mayaccount for the increased virus titer at 36 Camong certain strains (el Ridi et al. 1988).In contrast to increased titers at 36 C forthe H2N3 virus, H5N3 virus replicationwas less affected by temperature, althoughthe H5N3 virus also had the smallestincrease in titer. However, these data arenot unexpected as concomitant in vitro andin vivo findings in a murine model illustratethat H5N3 replication may occur at tem-peratures as low as 33 C (Hatta et al. 2007).

Embryonic organs, notably liver andkidney, of infected eggs were positive forboth genomic RNA and NP antigen, sug-gesting that infection and replication oc-curred within these organs. In contrast, themost-common sites of infection in avianspecies are the lower respiratory tract andthe gastrointestinal tract (Wright et al. 1992);therefore, we hypothesized that virus infec-tion and replication would occur in similartissues within embryonated alligators. How-ever, as we restricted our study to in vitroand in ovo infections, the tissue tropism forinfections in vivo remain unknown.

The viruses we used were all isolatedfrom birds and geographic regions thatoverlap with alligator habitats in NorthAmerica (Stallknecht et al. 1990). Thus, itis possible for these viruses to intersectwith alligator populations. The H5N3LPAI virus we tested was isolated from a

poultry farm in Texas (Lee et al. 2004) andreplicated in both primary cells andembryos at both low (33 C) and high(36 C) temperatures. An H5N1 HPAIvirus replicated in alligator cells in culture,and other HPAI viruses, have beenisolated from geographic regions overlap-ping with crocodilian habitats. While wedid not assess the infection and transmis-sion of influenza viruses in live crocodil-ians, the data suggest that alligators mightbe susceptible to influenza virus infectionand that further study is warranted.

ACKNOWLEDGMENTS

We extend a special thanks to Ruth Elsey ofthe Louisiana Department of Fish and Gamefor generously providing alligator eggs, to FrankMichel (University of Georgia, Athens, Geor-gia, USA) for technical support, Jon Yewdell(National Institute of Allergy and InfectiousDiseases, Bethesda, Maryland, USA) for pro-viding the mouse anti-influenza NP IgGhybridoma H16-L10, and to David Stallknecht(University of Georgia, Athens, Georgia, USA),David Suarez (Southeast Poultry ResearchLaboratory, US Department of Agriculture,Athens, Georgia, USA), and Richard Webby(Saint Jude Children’s Research Hospital,Memphis, Tennessee, USA) for providing theinfluenza viruses used in our study.

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Submitted for publication 10 August 2013.Accepted 7 August 2014.


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