Pathogenicity and Transmission of H5 and H7 Highly PathogenicAvian Influenza Viruses in Mallards

Mary J. Pantin-Jackwood,a Mar Costa-Hurtado,a* Eric Shepherd,a* Eric DeJesus,a Diane Smith,a Erica Spackman,a

Darrell R. Kapczynski,a David L. Suarez,a David E. Stallknecht,b David E. Swaynea

Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry Research Laboratory, U.S. National Poultry Research Center, Agricultural Research Service, U.S.Department of Agriculture, Athens, Georgia, USAa; Southeastern Cooperative Wildlife Disease Study, The University of Georgia, Athens, Georgia, USAb

ABSTRACT

Wild aquatic birds have been associated with the intercontinental spread of H5 subtype highly pathogenic avian influenza(HPAI) viruses of the A/goose/Guangdong/1/96 (Gs/GD) lineage during 2005, 2010, and 2014, but dispersion by wild waterfowlhas not been implicated with spread of other HPAI viruses. To better understand why Gs/GD H5 HPAI viruses infect and trans-mit more efficiently in waterfowl than other HPAI viruses, groups of mallard ducks were challenged with one of 14 different H5and H7 HPAI viruses, including a Gs/GD lineage H5N1 (clade 2.2) virus from Mongolia, part of the 2005 dispersion, and theH5N8 and H5N2 index HPAI viruses (clade 2.3.4.4) from the United States, part of the 2014 dispersion. All virus-inoculatedducks and contact exposed ducks became infected and shed moderate to high titers of the viruses, with the exception that mal-lards were resistant to Ck/Pennsylvania/83 and Ck/Queretaro/95 H5N2 HPAI virus infection. Clinical signs were only observedin ducks challenged with the H5N1 2005 virus, which all died, and with the H5N8 and H5N2 2014 viruses, which had decreasedweight gain and fever. These three viruses were also shed in higher titers by the ducks, which could facilitate virus transmissionand spread. This study highlights the possible role of wild waterfowl in the spread of HPAI viruses.

IMPORTANCE

The spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the Gs/GD lineage by migratory waterfowl is aserious concern for animal and public health. H5 and H7 HPAI viruses are considered to be adapted to gallinaceous species(chickens, turkeys, quail, etc.) and less likely to infect and transmit in wild ducks. In order to understand why this is differentwith certain Gs/GD lineage H5 HPAI viruses, we compared the pathogenicity and transmission of several H5 and H7 HPAI vi-ruses from previous poultry outbreaks to Gs/GD lineage H5 viruses, including H5N1 (clade 2.2), H5N8 and H5N2 (clade 2.3.4.4)viruses, in mallards as a representative wild duck species. Surprisingly, most HPAI viruses examined in this study replicated welland transmitted among mallards; however, the three Gs/GD lineage H5 HPAI viruses replicated to higher titers, which couldexplain the transmission of these viruses in susceptible wild duck populations.

Wild aquatic birds, especially of the orders Anseriformes(ducks, geese, and swans) and Charadriiformes (shorebirds,

gulls, terns, and auks) are the natural reservoirs of avian influenza(AI) viruses (1). These AI viruses are highly host adapted, typicallyreplicating in epithelial cells of the gastrointestinal tract and pro-ducing subclinical infections. Periodically, these AI viruses trans-mit from wild aquatic to domestic birds, producing subclinicalinfections or, occasionally, respiratory disease and drops in eggproduction, with such transmission and infections being mostpermissive for domestic waterfowl species (2). This virus pheno-type is termed low-pathogenicity or low-pathogenic (LP) AI virusand can be any combination of the 16 hemagglutinin (HA) and 9neuraminidase (NA) subtypes. However, a few H5 and H7 LPAIviruses after circulating in gallinaceous poultry (chickens, turkeys,quail, etc.) have mutated to produce the highly pathogenic (HP) phe-notype of AI viruses (3). These HPAI viruses cause severe systemicdisease and high mortality in gallinaceous poultry and are typicallyeasily transmissible among them (4). Historically, HPAI viruses havenot caused outbreaks or widespread infections in wild birds except forthe die-off that occurred among common terns (Sterna hirundo) inSouth Africa during 1961 (5). However, since 2002, the A/goose/Guangdong/1/96 (Gs/GD) lineage H5N1 HPAI viruses have causedinfections, illness, and death in a variety of captive, zoo, and wild birdspecies, including waterfowl (6–9).

In both wild and domestic ducks, experimental inoculationwith the Gs/GD lineage of H5N1 HPAI viruses can produce arange of clinical outcomes from asymptomatic infections to severedisease with mortality (reviewed in reference 10; see also refer-ences 11 to 18). Both sick and asymptomatic infected ducks shedhigh virus quantities, increasing the risk of transmission. Further-more, migratory waterfowl have been infected with the Gs/GDlineage of H5N1 HPAI viruses in the field and, based on field

Received 14 June 2016 Accepted 19 August 2016

Accepted manuscript posted online 24 August 2016

Citation Pantin-Jackwood MJ, Costa-Hurtado M, Shepherd E, DeJesus E, Smith D,Spackman E, Kapczynski DR, Suarez DL, Stallknecht DE, Swayne DE. 2016.Pathogenicity and transmission of H5 and H7 highly pathogenic avian influenzaviruses in mallards. J Virol 90:9967–9982. doi:10.1128/JVI.01165-16.

Editor: S. Schultz-Cherry, St. Jude Children’s Research Hospital

Address correspondence to Mary J. Pantin-Jackwood,Mary.Pantin-Jackwood@ars.usda.gov.

* Present address: Mar Costa-Hurtado, Research Group on Infectious Diseases inProduction Animals and Swine and Poultry Infectious Diseases Research Center,Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, Quebec,Canada; Eric Shepherd, Poultry Diagnostic and Research Center, College ofVeterinary Medicine, The University of Georgia, Athens, Georgia, USA.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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epidemiology, such birds have contributed to long-distance virusspread, including intercontinentally, during three distinct timeperiods: 2005, 2010, and 2014 (18–28). Experimental studies insome wild duck species, especially mallards (Anas platyrhynchos),have confirmed their potential to be long-distance vectors of theseviruses (17, 18).

As the Gs/GD lineage H5N1 viruses continue to circulate andspread, the HA genes have diversified into multiple genetic lin-eages or clades. In 2005, an H5N1 virus of the Gs/GD lineage wasisolated from outbreaks with mortality in several waterfowl spe-cies in China (7). This virus lineage, defined as clade 2.2, movedthrough migratory birds and often was transmitted to poultry,resulting in outbreaks in more than 20 countries (25). A secondspillover event from poultry to wild birds of a different H5N1virus, clade 2.3.2.1, began appearing in wild birds in late 2007, andthis lineage of virus, although not spreading as far geographically,resulted in poultry outbreaks in at least seven different countriesin Asia. This lineage of virus has been detected in wild birdsthrough 2012 (26, 27). Viruses from this clade 2.3.2.1 probablyspread through wild birds to several Asian countries in 2008 and toEurope in 2010 (28). Since 2008, subclade 2.3.4.4 has reassortedwith multiple neuraminidase subtypes to form widely circulatingH5N2, H5N3, H5N5, H5N6, and H5N8 subtypes of HPAI viruses(22, 29–32). In early 2014, outbreaks of a reassortant H5N8 HPAIvirus were reported in South Korea in poultry and wild aquaticbirds (33), with migratory aquatic birds strongly suspected inplaying a key role in the spread of the virus (23). In late autumn2014, H5N8 HPAI viruses were detected in Siberia, several coun-tries in Europe, in South Korea, and in Japan (22, 31). Concur-rently, this virus was detected in the United States in captive fal-cons, wild birds, and backyard aquatic and gallinaceous poultry(34). In addition, another novel reassortant HPAI virus of H5clade 2.3.4.4 (H5N2) was identified as the cause of an outbreak inpoultry farms in British Columbia, Canada (35), and was subse-quently detected in the United States in wild waterfowl and back-yard poultry. From March to June 2015, the H5N2 reassortantvirus was found in wild aquatic birds, raptors, and backyard andcommercial poultry flocks in the Midwestern region of the UnitedStates (36). This reassortant H5N2 virus predominated in theUnited States, and extensive farm-to-farm transmissions occurredin the Midwestern region. Over 7.5 million turkeys and 42.1 mil-lion chickens died or were culled during this outbreak whichended in June 2015 (37).

Although data are limited and reported results are mixed, mostH5/H7 HPAI viruses are considered to be adapted to gallinaceousspecies and therefore less likely to infect, replicate efficiently, andcause disease in domestic or wild ducks (38–40). Experimentallyor naturally, mortality in domestic ducks caused by HPAI viruseshad been infrequently reported before the Gs/GD H5N1 HPAIoutbreaks in Asia (10, 38, 41). Similarly, infections and diseasefrom HPAI viruses in domestic ducks have been rare. Wild aquaticbirds are the genetic reservoirs of LPAI viruses but are not geneticor long-term reservoirs of HPAI viruses (42). In most experimen-tal studies, ducks intranasally (i.n.) inoculated with H5 or H7HPAI viruses showed very mild or no clinical signs (40, 43–48),but virus was isolated in some cases from tracheal and cloacalswabs (44, 47, 48) and recovered from the trachea, gut, liver, brain,and spleen (40). Based on the results of these studies, it is not clearwhether domestic or wild ducks can easily become infected with

HPAI viruses other than the Gs/GD lineage and transmit the virusto naive ducks.

Recurring outbreaks of H5 and H7 HPAI in poultry and therecent outbreaks of H5N8 and H5N2 HPAI underscore the needto better understand the pathogenesis and transmission of theseviruses in wild birds. The goal of the present study was to describethe pathogenicity, viral shedding patterns, and transmissibility ofNorth American clade 2.3.4.4 H5 viruses in mallards and to deter-mine whether they differ from other Gs/GD lineage viruses andhistoric H5 and H7 HPAI viruses. In this study, we used mallardsas a model system since they have been naturally infected withGs/GD H5 viruses in both Eurasia and North America, they rep-resent the waterfowl species most utilized in experimental infec-tions, and they are closely related to the most commonly reareddomestic duck species.

MATERIALS AND METHODSViruses. The following 14 HPAI viruses were used in this study: Gs/GD lineage, clade 2.3.4.4 —A/Northern pintail/Washington/40964/2014(H5N2) (Np/WA/14) and A/Gyrfalcon/Washington/40188-6/2014(H5N8) (Gf/WA/14), and clade 2.2—A/Whooper swan/Mongolia/244/2005 (H5N1) (Ws/Mongolia/05); A/chicken/Chile/184240-1/2002(H7N3) (Ck/Chile/02), A/chicken/Canada/314514-2/2005 (H7N3) (Ck/Canada/05), A/chicken/Jalisco/CPA1/2012 (H7N3) (Ck/Jalisco/12),A/chicken/Victoria/85 (H7N4 (Ck/Victoria/85), A/chicken/North Korea/7916/2005 (H7N7) (Ck/North Korea/05); A/chicken/Netherlands/1/2003(H7N7) (Ck/Netherlands/03), A/turkey/Italy/4580/99 (H7N1) (Tk/Italy/99), 9) A/chicken/Pennsylvania/1370/83 (H5N2) (Ck/PA/83), A/chicken/Queretaro/14588-19/95 (H5N2) (Ck/Queretaro/95), A/turkey/Ireland/1378/83 (H5N8) (Tk/Ireland/83), and A/tern/South Africa/61 (H5N3)(Tern/South Africa/61). Three LPAI viruses were included as controls:A/mallard/Minnesota/410/2000 (H5N2) (Ml/MN/00), A/mallard/Ohio/421/87 (H7N8) (Ml/OH/87), and A/mallard/Sweden/85/2002 (H7N2)(Ml/Sweden/85). Viruses were obtained from the Southeast Poultry Re-search Laboratory repository. The isolates used were the earliest pass ineggs available for each virus. The viruses were titrated by allantoic sacinoculation of 9- to 10-day-old embryonated chicken eggs (ECEs) accord-ing to standard procedures (49). Allantoic fluid was diluted in brain heartinfusion (BHI) medium (BD Bioscience, Sparks, MD) in order to obtainan inoculum with 106 50% egg infectious dose (EID50) per 0.1 ml/bird. Allchallenge doses were confirmed by retitrating the inocula in ECEs.

Birds. Mallards were obtained at 1 day of age from a commercialhatchery. Serum samples were collected from 15 ducks per experiment toascertain that the birds were serologically negative for AI viruses by block-ing ELISA (FlockCheck avian influenza MultiS-Screen antibody test;IDEXX Laboratories, Westbrook, ME). At 2 weeks of age, the ducks werehoused in self-contained isolation units ventilated under negative pres-sure with inlet and exhaust HEPA (high-efficiency particulate arres-tance)-filtered air and maintained under continuous lighting. Feed andwater were provided with ad libitum access. All procedures were per-formed according to the requirements of protocols approved by the Insti-tutional Animal Care and Use Committee and the Institutional BiosafetyCommittee.

Experimental design. Two identical experiments were conducted. Inexperiment 1, the ducks were inoculated with Np/WA/14 (H5N2), Gf/WA/14 (H5N8), or Ws/Mg/05 (H5N1) HPAI viruses (Table 1). In addi-tion, a H5 North American lineage LPAI virus Ml/MN/00 H5N2 wasincluded for comparison purposes. In experiment 2, seven H7 HPAI vi-ruses and four H5 HPAI viruses were examined, as well as two H7 LPAIviruses for comparison (Table 1). In both experiments, the ducks wereseparated into a sham-inoculated control group and virus-inoculatedgroups. Ducks were i.n. inoculated, via choanal cleft, with 106 EID50/0.1ml of each virus. Sham-inoculated control ducks were i.n. inoculated with0.1 ml of sterile allantoic fluid diluted 1:300 in BHI. Three naive ducks

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were added to each group at 1 day postinoculation (dpi) to examine forvirus transmission. Body temperatures were taken from all ducks at 2 and4 dpi, and body weights were determined at 2, 4, 7, and 14 dpi. Two ducksfrom each group were euthanized at 4 dpi to examine for gross lesions, andportions of the brain, lung, spleen, skeletal muscle, and heart (first exper-iment) and of the brain, lung, and spleen (second experiment) were col-lected frozen for subsequent virus detection. In the first study, a full set oftissue samples was also collected for microscopic evaluation as well (nasalcavity, trachea, lungs, air sacs, eye lid, brain, spleen, liver, intestine, pan-creas, kidney, adrenal glands, Harderian glands, thymus, bursa, heart, andskeletal muscle). Tissues were fixed in 10% neutral buffered formalinsolution, paraffin embedded, sectioned, and stained with hematoxylinand eosin. Duplicate sections were stained by immunohistochemicalmethods to determine the influenza viral antigen distribution in individ-ual tissues (50). The remaining ducks were observed for clinical signs overa 14-day period during which time clinical signs were recorded. Ducksthat showed severe neurological signs, stopped eating or drinking, or re-mained recumbent were euthanized and were reported as dead on thenext day for the calculation of mean death times. Oropharyngeal (OP) andcloacal (CL) swabs were collected at 2, 4, 7, 11, and 14 dpi from directlyinoculated birds, and at 1, 3, 6, 10, and 13 days after contact from contact-exposed birds to determine virus shedding. Surviving ducks were bled at14 dpi for serology and euthanized by the intravenous administration ofsodium pentobarbital (100 mg/kg [body weight]).

Virus quantifications. OP and CL swabs were collected in 1 ml of BHIbroth with a final concentration of gentamicin (1,000 �g/ml), penicillin G(10,000 U/ml), and amphotericin B (20 IU/ml) and kept frozen at �70°Cuntil processed. Viral RNA was extracted using a MagMAX AI/ND viralRNA isolation kit (Ambion, Austin, TX). Quantitative real-time reversetranscription-PCR (qRT-PCR) for AIV detection was performed as pre-viously described (32). qRT-PCRs targeting the influenza virus M gene(51) were conducted by using a AgPath-ID one-step RT-PCR kit (Am-bion) and the ABI 7500 Fast real-time PCR system (Applied Biosystems,Carlsbad, CA). The RT step conditions were 10 min at 45°C and 10 min at

95°C. The cycling conditions were 45 cycles of 15 s at 95°C and 45 s at60°C. Virus titers in frozen tissue samples were determined by weighing,homogenizing, and diluting tissues in BHI to a 10% (wt/vol) concentra-tion. Viral RNA was extracted using TRIzol LS reagent (Invitrogen, Carls-bad, CA) and a Qiagen RNeasy minikit (Qiagen). Equal amounts of RNAextracted from the tissue samples were used in the qRT-PCR assay (50ng/�l). For virus quantification, a standard curve was established withRNA extracted from dilutions of the same titrated stock of the challengevirus, and the results are also reported as EID50/ml or EID50/g equivalents.The calculated qRT-PCR lower detection limit for the viruses varied be-tween 101.6 and 101.9 EID50/ml.

Serology. Hemagglutination inhibition (HI) assays using homolo-gous antigens were performed to quantify antibody responses to virusinfection with serum collected from ducks at 14 dpi as previously de-scribed (52). HI titers were reported as reciprocal log2 titers, with a 3-log2

(a titer of 1:8) titer or greater considered positive.Statistical analyses. One-way analysis of variance, along with Tukey’s

multiple-comparison tests, was applied to analyze body weights, bodytemperatures, and titers of virus shedding at 4 dpi using Prism v.5.01software (GraphPad, La Jolla, CA). A P value of 0.05 was considered sig-nificant.

RESULTSClinical signs, body temperature, and body weights in mallardsinfected with HPAI and LPAI viruses. No clinical signs or mor-tality was observed in mallards inoculated with any LPAI or HPAIviruses in both experiments, with the exception of those inocu-lated with H5 Gs/GD HPAI viruses. In experiment 1, ducks inoc-ulated with Ws/Mongolia/05 (H5N1) presented with lethargy, an-orexia, and neurological signs, and all of them died by 4 dpi,similar to previous reports with some Gs/GD lineage H5N1 HPAIvirus infections in ducks (10). Ducks inoculated with the Gf/WA/14 (H5N8) or Ws/Mongolia/05 (H5N1) HPAI viruses had

TABLE 1 Body temperatures and body weights of mallards inoculated with H5 and H7 LPAI and HPAI viruses

Expt and virus

Avg � SEMa

Body temp (oF) Body wt (kg)

2 dpi 4 dpi 2 dpi 4 dpi 7 dpi 14 dpi

Expt 1Controls 107.4 � 0.3 107.4 � 0.2 0.34 � 0.01 0.39 � 0.01 0.48 � 0.01 0.64 � 0.02Ml/MN/00 (H5N2) LPAIV 107.8 � 0.2 107.8 � 0.3 0.34 � 0.001 0.40 � 0.01 0.48 � 0.02 0.65 � 0.02Np/WA/14 (H5N2) HPAIV 107.1 � 0.2 107 � 0.2 0.29 � 0.001* 0.32 � 0.01** 0.40 � 0.02* 0.57 � 0.03Gf/WA/14 (H5N1) HPAIV 108.9 � 0.2*** 107.8 � 0.5 0.29 � 0.01* 0.34 � 0.01* 0.44 � 0.02 0.64 � 0.03Ws/Mongolia/05 (H5N1) HPAIV 109 � 0.2*** ND 0.27 � 0.001*** ND ND ND

Expt 2Controls 107.2 � 0.2 107.2 � 0.1 0.30 � 0.01 0.35 � 0.01 0.44 � 0.1 0.60 � 0.02Ml/OH/87 (H7N8) LPAIV 108.8 � 0.1 107.7 � 0.2 0.30 � 0.01 0.34 � 0.01 0.43 � 0.01 0.62 � 0.01Ml/Sweden/02 (H7N7) LPAIV 107 � 0.3 107 � 0.2 0.28 � 0.02 0.36 � 0.02 0.41 � 0.02 0.60 � 0.02Ck/Chile/02 (H7N3) HPAIV 106.9 � 0.3 107.9 � 0.2 0.26 � 0.01 0.29 � 0.01* 0.42 � 0.01 0.56 � 0.01Ck/Canada/05 (H7N3) HPAIV 107.8 � 0.1 108 � 0.3 0.27 � 0.01 0.33 � 0.01 0.43 � 0.02 0.53 � 0.02Ck/Jalisco/12 (H7N3) HPAIV 107.6 � 0.1 107.4 � 0.2 0.25 � 0.02 0.30 � 0.02 0.40 � 0.02 0.57 � 0.02Ck/Victoria/85 (H7N7) HPAIV 106.7 � 0.3 107.1 � 0.3 0.26 � 0.01 0.31 � 0.01 0.38 � 0.01 0.49 � 0.02*Ck/North Korea/05 (H7N7) HPAIV 107.5 � 0.1 107 � 0.3 0.26 � 0.01 0.32 � 0.01 0.40 � 0.01 0.53 � 0.02Ck/Netherlands/03 (H7N7) HPAIV 107.3 � 0.1 107.7 � 0.2 0.29 � 0.01 0.33 � 0.01 0.41 � 0.02 0.59 � 0.03Tk/Italy/99 (H7N1) HPAIV 107 � 0.2 107.9 � 0.2 0.29 � 0.01 0.34 � 0.001 0.42 � 0.02 0.59 � 0.02Ck/PA/83 (H5N2) HPAIV 106.8 � 0.1 106.4 � 0.3 0.27 � 0.01 0.31 � 0.02 0.40 � 0.02 0.55 � 0.02Ck/Queretaro/95 (H5N2) HPAIV 107.2 � 0.2 107.3 � 0.3 0.25 � 0.01* 0.30 � 0.02 0.38 � 0.02 0.53 � 0.02Tk/Ireland/83 (H5N8) HPAIV 106.5 � 0.2 106.4 � 0.2 0.27 � 0.01 0.32 � 0.001 0.41 � 0.01 0.58 � 0.01Tern/South Africa/61 H5N3 HPAIV 107.4 � 0.2 106.9 � 0.2 0.28 � 0.02 0.33 � 0.01 0.41 � 0.02 0.56 � 0.02

a Significant differences in body temperatures or body weights when comparing groups in each experiment are indicated by asterisks (*, P � 0.01; **, P � 0.001; ***, P � 0.0001).ND, not done.

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significantly higher body temperatures at 2 dpi than ducks inocu-lated with Np/WA/14 (H5N2) HPAI virus or the Ml/MN/00 LPAIvirus or the sham-inoculated control ducks (P � 0.0001) (Table1). Three ducks inoculated with the H5N8 virus still had fever at 4

dpi (Fig. 1). Ducks inoculated with the Np/WA/14 (H5N2), Gf/WA/14 (H5N8), and Ws/Mongolia/05 (H5N1) HPAI viruses hadsignificantly lower body weights than control ducks and ducksinoculated with the LPAI virus at 2 and 4 dpi (Table 1 and Fig. 2)

FIG 1 Experiment 1: mean body temperatures of mallards inoculated with LPAI and HPAI viruses at 2 and 4 dpi. Bars represent the standard deviations of themean. Significant differences in body temperature compared to controls are indicated with asterisks (***, P � 0.0001).

FIG 2 Experiment 1: mean body weights of mallards inoculated with LPAI and HPAI viruses at 2, 4, 7, and 14 dpi. Bars represent the standard deviations of themean. Significant differences in body weight compared to controls are indicated with asterisks (*, P � 0.01; **, P � 0.001; ***, P � 0.0001).

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(P � 0.01 to P � 0.0001). Np/WA/14 (H5N2)-infected ducks alsohad significantly lower weights at 7 dpi (P � 0.01). No differencesin body weights were observed between groups at 14 dpi.

In experiment 2, although there were differences in body tem-peratures at 2 and 4 dpi in ducks inoculated with the HPAI virusesand the LPAI viruses compared to controls, these differences werenot significant (Table 1 and Fig. 3). There was also no significanteffect of AI virus infection on body weights when examined at 2, 4, 7,and 14 dpi compared to sham-inoculated controls for most of thegroups (Table 1 and Fig. 4). Ducks infected with Ck/Queretaro/95(H5N2), Ck/Chile/02 (H7N3), and Ck/Victoria/85 (H7N7) hadlower body weights than control ducks, but at only one time point (2,4, and 14 dpi, respectively; P � 0.01).

Lesions induced by HPAIV infections in mallards. No grosslesions were observed in any of the sham-inoculated control ducksnecropsied at 4 dpi in experiments 1 and 2. In experiment 1, nolesions were observed in mallards inoculated with the LPAI virus.Gross lesions were observed in all six ducks inoculated with the H5HPAI viruses, including mild to moderate dehydration, emptyintestines, splenomegaly, and thymus atrophy. Also, nasal dis-charge, cyanotic bill and toes, dilated and flaccid heart with in-creased pericardial fluid, renal pallor, and congested brain wereobserved in ducks inoculated with Ws/Mongolia/05 (H5N1). No

gross lesions were observed in any of the ducks necropsied inexperiment 2.

Tissues collected from ducks in experiment 1 were examinedfor microscopic lesions. Ducks inoculated with Ml/MN/00(H5N2) developed lesions consistent with LPAI virus infection;these were mostly confined to the upper respiratory tract. Mildcatarrhal rhinitis and sinusitis, with mucocellular exudates con-taining sloughed epithelial cells, submucosal edema, and glandu-lar hyperplasia, were observed. The trachea showed mild degen-erative changes of the overlying epithelium and mild lymphocyticinfiltration in the submucosa and mild edema. Lesions in the gas-trointestinal tract consisted of mild proliferation of gut-associatedlymphoid tissues. The remaining organs did not show significanthistopathologic changes. In contrast, severe and widespread mi-croscopic lesions were observed in tissues from ducks inoculatedwith Ws/Mongolia/05 (H5N1); these changes were similar tothose observed with other Gs/GD lineage H5N1 HPAI viruses(10). The most consistent lesions were moderate to severe rhinitisand sinusitis, moderate tracheitis and bronchitis, mild to moder-ate interstitial pneumonia, airsacculitis, and moderate multifocalnecrosis of cardiac myofibers and, in the brain, randomly scat-tered foci of malacia with gliosis. Also commonly observed weremultifocal pancreatitis, necrosis of the epithelia of the Harderianglands, and moderate multifocal areas of vacuolar degeneration tonecrosis of the corticotrophic cells of the adrenal gland. Mild tomoderate necrosis of hepatocytes with sinusoidal histiocytosis wasfound in the liver. The spleen, thymus, bursa, and mucosa-asso-ciated lymphoid tissue showed moderate to severe lymphoid de-pletion. Ducks inoculated with the H5N8 and H5N2 HPAI viruseshad moderate rhinitis, sinusitis, tracheitis, bronchitis, mild inter-stitial pneumonia, and airsacculitis, and mild pancreatitis. Mildnecrosis of the epithelia of the Harderian glands was observed intwo ducks. The liver of one duck had lymphocytic infiltrations andfocal hepatocyte necrosis. Another duck had focal pancreatitis.Mild to moderate lymphoid depletion was observed in all lym-phoid organs.

In order to determine sites of virus replication, immunohisto-chemical staining for AI virus antigen was conducted. In ducksinoculated with the LPAI virus, viral antigen staining was onlyobserved in nasal and trachea epithelial cells (Table 2). Viral anti-gen staining was present in multiple tissues from ducks infectedwith the H5 HPAI viruses, indicating systemic infection, but virusstaining was more widespread in tissues from ducks inoculatedwith Ws/Mongolia/05 (H5N1). Viral antigen was present in epi-thelial cells and macrophages in the nasal turbinates, trachea,lung, air sac, and Harderian and nasal glands, in pancreatic acinarcells, and in resident and infiltrating phagocytes of the thymus andspleen (Fig. 5). In addition, in ducks inoculated with Ws/Mongo-lia/05 (H5N1), viral antigen was also identified in the autonomicganglia of the enteric tract, feather epidermal cells, neurons andglial cells of the brain, hepatocytes and Kupffer cells in the liver,fragmented cardiac and skeletal myofibers, and adrenal cortico-trophic cells (Fig. 5). In one duck inoculated with Np/WA/14(H5N2), viral staining was also present in the neuron and ependy-mal cells in the brain, and in ducks inoculated with Gf/WA/14(H5N8) we found viral staining in phagocytes in the eyelid sub-mucosa and in hepatocytes.

Viral RNA quantification in swabs and tissues. Oropharyn-geal (OP) and cloacal (CL) viral shedding was examined by qRT-PCR, and the results are shown in Table 3 and Fig. 6, 7, 8, and 9. All

FIG 3 Experiment 2: mean body temperatures of mallards inoculated withLPAI and HPAI viruses at 2 and 4 dpi. Bars represent the standard deviations ofthe mean. No significant differences in body temperature among groups weredetected.

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virus-inoculated ducks became infected, as determined by the de-tection of viral RNA in swabs, with the exception of three ducksinoculated with Ck/PA/83 (H5N2) HPAI and four ducks inocu-lated with Ck/Queretaro/95 (H5N2) (Table 3). In experiment 1,mallards inoculated with Ws/Mongolia/05 (H5N1) shed high ti-ters of virus by the OP route at 2 and 4 dpi (105.7 to 107.8 EID50/ml)(Fig. 6). Mallards inoculated with Np/WA/14 (H5N2) or Gf/WA/14 (H5N8) shed moderate to high titers of virus at 2 and 4 dpi(103.8 to 106.7 EID50/ml). Mallards inoculated with the Ml/MN/00(H5N2) LPAI virus shed 101.4 to 105.1 EID50/ml by the OP route atthese same time points, and there was more variability amongtiters at 2 dpi compared than that observed with the HPAI viruses.In ducks inoculated with the HPAI viruses, CL virus titers werealmost 2 log10 lower than what was observed in OP swabs at 2 and4 dpi. The duration of virus shedding varied among the virusesexamined. Most of the ducks inoculated with Gf/WA/14 (H5N8)virus shed low titers, or no virus, by 11 dpi, and most of the mal-lards inoculated with Np/WA/14 (H5N2) virus or the LPAI virusshed virus up to 14 dpi (Fig. 6). Ducks inoculated with the Ws/Mongolia/05 (H5N1) virus were dead by 4 dpi.

In experiment 2, mallards inoculated with the H7 LPAI virusesshed virus by both the OP and CL routes, but shedding occurredfor a longer period by the CL route (Fig. 7 and 8). For all H7viruses, the CL titers at 2 dpi were lower than the OP titers, whichis in part explained by the route of virus inoculation, with the

viruses initially replicating in the upper respiratory tract and laterin the intestine (Fig. 7 and 8). The highest virus titers were de-tected at 2 and 4 dpi. The pattern of virus shedding was similarbetween groups of ducks inoculated with Ck/Chile/02 (H7N3)and Ck/Canada/05 (H7N3) HPAI viruses, with most ducks shed-ding up to 7 dpi by the OP route and until 11 dpi by the CL route(Fig. 7). The Ck/Jalisco/12 (H7N3), Ck/Victoria/85 (H7N7), Ck/North Korea/05 (H7N7), Ck/Netherlands/03 (H7N7), and Tk/Italy/05 (H7N1) HPAI viruses were similarly shed up to 11 dpi byboth routes (Fig. 8). Viral RNA was detected in swabs from someindividual ducks at 14 dpi in almost all groups.

Regarding the H5 HPAI viruses, not all mallards inoculatedwith Ck/PA/83 (H5N2) or Ck/Queretaro/95 (H5N2) becameinfected, with only 7 or 6 of 10 mallards shedding the respectiveviruses (Fig. 9). Although more virus was shed by the OP routethan the CL route, the titers were low in both groups, and mostducks only shed until 4 dpi. Mallards inoculated with Tk/Ireland/83 (H5N8) shed mostly by the OP route with someducks shedding high titers at 4 dpi (Fig. 9). The Tern/SouthAfrica/61 (H5N3) HPAI virus was shed at moderate titers byboth routes (Fig. 9).

When comparing OP virus titers at 4 dpi in all groups (exper-iments 1 and 2 combined), significantly higher titers were shed byducks inoculated with Ws/Mongolia/05 (H5N1), Np/WA/14(H5N2), and Gf/WA/14 (H5N8) (P � 0.1 to 0.0001) than the rest

FIG 4 Experiment 2: mean body weights of mallards inoculated with LPAI and HPAI viruses at 2, 4, 7, and 14 dpi. Bars represent the standard deviations of themean. Significant differences in body weight compared to controls are indicated with asterisks (*, P � 0.01).

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of the groups, with no significant difference among the three (Ta-ble 3). Ducks inoculated with Ck/PA/83 (H5N2) shed significantlysmaller amounts of virus than ducks in all other groups, with theexception of Ck/Queretaro/95 (H5N2). Ducks inoculated withCk/Queretaro/95 (H5N2) also shed less virus than ducks inocu-lated with Ml/OH/87 (H5N2), Ck/North Korea/05 (H7N7), Ck/Canada/05 (H7N3), Tk/Italy/99 (H7N1), Ck/Victoria/85 (H7N7),and Tern/South Africa/61 (H5N3). With some minor exceptions,there were no significant differences in the titers of virus shed atthis time point among the rest of the groups.

The presence of viral RNA was also examined in tissues col-lected at 4 dpi from virus-inoculated mallards (Table 4). In exper-iment 1, lung, spleen, brain, heart and muscle were examined; inexperiment 2, only the lungs, spleen, and brain were examined.Low viral titers, or titers under the limit of detection, were ob-

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FIG 5 Immunohistochemical staining for AIV antigen in tissues of mallardsinfected with Gs/GD H5 lineage HPAI viruses. Tissues were collected at 4 dpi.Virus antigen is stained red. Panels A, B, C, D, and E show tissues from ducksinfected with Gf/WA/14 (H5N8) HPAIV; panels F, G, and H show tissues fromducks infected with Ws/Mongolia/05 (H5N1) HPAIV. (A) Trachea; (B) air sac;(C) liver; (D) Harderian gland; (E) nasal gland; (F) pancreas; (G) cerebellum;(H) heart. Magnification, �40. Viral antigen was present in epithelial cells andmacrophages in the trachea, air sac, Harderian and nasal glands, in hepatocytesand Kupffer cells in the liver, in pancreatic acinar cells, in neurons and glialcells of the brain, and in cardiac myofibers.

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served in most tissues from LPAI virus-inoculated groups. In ex-periment 1, the titers were consistently high in tissues from ducksinoculated with Ws/Mongolia/05 (H5N1). High titers were alsoobserved in some of the tissues from ducks inoculated with theH5 North American Gs/GD lineage HPAI viruses. In general,tissue titers were higher in these two groups than in all otherHPAI virus-inoculated groups in experiment 2, with the excep-tion of Ck/Chile/02 (H7N3), Ck/North Korea/05 (H7N7), andTk/Italy/99 (H7N1), which also had high virus titers in sometissues (Table 4).

Serology. When examined at 14 dpi, most ducks had detect-able antibody titers against the viruses (Table 3). Serology is notavailable for the group inoculated with the Ws/Mongolia/05(H5N1) HPAI virus since all of the ducks died.

Transmission to contacts. To assess virus transmission, naiveducks were added to the experimental groups at 1 dpi. All contactexposure ducks became infected with AIV, as demonstrated byvirus shedding and/or seroconversion, with the exception of thosein the groups challenged with Ck/Queretaro/95 (H5N2) andTern/South Africa/61 (H5N3) in which two and one contactducks, respectively, did not become infected (Table 3).

DISCUSSION

In this study, we compared the pathobiology and transmission of14 HPAI viruses, including both H5 and H7 viruses in mallards, toassess the potential role of this model waterfowl species in dissem-inating HPAI viruses. Many studies have examined the pathoge-nicity of Gs/GD H5N1 HPAI viruses in domestic duck species andcaptive reared mallards (reviewed in reference 10), but only a lim-ited number of studies have investigated the pathogenicity ofother H5 and H7 HPAI viruses in domestic ducks species (38, 40,43–46, 53, 54) and in nondomestic ducks (17, 18, 47, 48, 55, 56). In

most of these studies, virus infection, shedding, and transmissionwere not thoroughly examined. With the exception of ducks in-oculated with A/fowl/Germany/34-Rostock H7N1 (38), clinicalsigns or mortality were not observed with domestic ducks. How-ever, neurological signs and deaths were reported in Muscovyducks following infections with a H7N1 HPAI virus during the1999-2000 outbreaks in Italy (41), and H5N2 HPAI viruses weredetected in wild waterfowl in Nigeria (56). All of the HPAI virusesexamined in our study infected the mallards and transmitted to directcontacts. Two viruses, Ck/PA/83 (H5N2) and Ck/Queretaro/95(H5N2), failed to infect all ducks after direct challenge, and twoviruses, CK/Queretaro/95 and Tern/South Africa/61 (H5N3),failed to infect all of the contact ducks. Clinical signs were onlyobserved in ducks challenged with Gs/GD lineage H5 HPAI vi-ruses; more specifically, the Ws/Mongolia/05 (H5N1) virus killedall mallards, the Np/WA/14 (H5N2) and Gf/WA/14 (H5N8) vi-ruses affected weight, and the Gf/WA/14 virus induced fever. Thehigh mortality observed in the mallards infected with Ws/Mongo-lia/05 (H5N1) has also been reported with many other Gs/GDlineage H5N1 HPAI viruses in both domestic and nondomesticducks, but not all viruses from this lineage cause mortality (10).Although the Ws/Mongolia/05 (H5N1) virus belongs to the clade2.2 H5N1 viruses that spread from Asia into Europe in 2005, it isnot representative of all viruses from this clade. Viruses from clade2.2 were reported to cause mortality in wild waterfowl (6, 7, 57),but differences in pathogenicity have been described between vi-ruses examined experimentally in domestic ducks (53, 57–61),indicating that ducks could become infected with these virusesand not necessarily show clinical signs but still shed large amountsof virus and transmit virus efficiently.

In ducks, the naturally occurring endemic LPAI viruses aretypically enterotropic and are shed primarily through feces (62–64).

TABLE 3 Infection, virus shed titers, and seroconversion of ducks inoculated with LPAI and HPAI viruses and contact-exposed ducksa

Expt and virus

Direct inoculates Contact-exposed ducks

No. ofinfectedducks/totalducks

No. ofdaysviruspositive

Oropharyngealshed titer(mean at4 dpi)

Cloacal shedtiter (meanat 4 dpi)

No. ofantibody-positiveducks/total ducks(titer range)

No. ofinfectedducks/totalducks

Oropharyngealshed titer(mean at 3days aftercontact)

Cloacal shedtiter (mean at3 days aftercontact)

No. ofantibody-positiveducks/total ducks(titer range)

Expt 1Ml/MN/00 (H5N2) LPAIV 10/10 �14 3.8 3.9 4/8 (8–64) 3/3 4.3 4.0 1/3 (8)Np/WA/14 (H5N2) HPAIV 10/10 �14 5.8 3.7 8/8 (16–32) 3/3 5.5 4.0 3/3 (16)Gf/WA/14 (H5N8) HPAIV 10/10 �14 5.7 3.8 7/7 (8–64) 3/3 6.1 3.6 2/3 (16)Ws/Mongolia/05 (H5N1) HPAIV 10/10 ND 6.6 5.0 ND 3/3 6.3 4.7 ND

Expt 2Ml/OH/87 (H7N8) LPAIV 10/10 �14 4.8 4.6 8/8 (16–64) 3/3 4.3 4.8 3/3 (16)Ml/Sweden/02 (H7N7) LPAIV 10/10 �14 4.1 5.3 8/8 (8–128) 3/3 4.0 5.3 3/3 (16–32)Ck/Chile/02 (H7N3) HPAIV 10/10 �14 4.0 4.1 8/8 (8–64) 3/3 2.8 3.5 3/3 (16–32)Ck/Canada/05 (H7N3) HPAIV 10/10 �14 4.3 5.1 8/8 (16–256) 3/3 3.8 5.6 3/3 (16–64)Ck/Jalisco/12 (H7N3) HPAIV 10/10 �14 4.1 5.3 8/8 (16–128) 3/3 4.4 5.0 3/3 (16–64)Ck/Victoria/85 (H7N7) HPAIV 10/10 �14 4.2 4.0 8/8 (16–64) 3/3 5.3 4.3 3/3 (8–128)Ck/North Korea/05 (H7N7) HPAIV 10/10 �14 4.5 3.9 8/8 (8–32) 3/3 4.9 3.3 3/3 (16)Ck/Netherlands/03 (H7N7) HPAIV 10/10 �14 3.7 3.1 8/8 (8–64) 3/3 3.5 3.8 3/3 (32–64)Tk/Italy/99 (H7N1) HPAIV 10/10 �14 4.4 3.0 8/8 (16–64) 3/3 2.8 2.9 3/3 (16)Ck/PA/83 (H5N2) HPAIV 7/10 �7 1.8 1.8 6/8 (8) 3/3 1.5 1.7 3/3 (8)Ck/Queretaro/95 (H5N2) HPAIV 6/10 �7 2.7 1.5 6/8 (8–127) 1/3 – – 1/3 (8)Tk/Ireland/83 (H5N8) HPAIV 10/10 �11 4.9 3.1 8/8 (256–624) 3/3 2.9 – 2/3 (8–64)Tern/South Africa/61 (H5N3) HPAIV 10/10 �14 4.2 3.0 8/8 (16–64) 2/3 – 3.3 1/3 (8)

a Two-week-old mallard ducks were i.n. inoculated with 106 EID50 of each virus. Ducks were considered virus positive if RNA was detected at 4 dpi. Virus titers were determined byquantitative real-time RT-PCR and are expressed as the log10 EID50/ml. The mean HI titers (log2) in ducks are indicated, with the range given in parentheses. The number of birdswith positive HI titers is shown (� threshold of positivity/total number of sera tested). –, negative; ND, not done.

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When wild waterfowl LPAI viruses jump to and become adaptedto gallinaceous species, the virus typically changes to becomemore respiratory-tropic with smaller amounts of detectable virusin feces. When these gallinaceous adapted viruses infect ducks, thevirus usually retains the respiratory-tropic replication pattern (65,66). In our study, the LPAI viruses examined were shed for longer

by the CL route than the OP route, although moderate replicationin the upper respiratory tract still occurred. As for the HPAI vi-ruses, different patterns of virus shedding were observed. The Ck/PA/83 (H5N2), Ck/Queretaro/95 (H5N2), and TK/Ireland/83(H5N8) viruses were shed at low titers and for fewer than 7 days,with minimal CL shedding, indicating adaptation to gallinaceous

FIG 6 Mean viral shedding in mallards inoculated with Ml/MN/00 (H5N2) LPAI, Np/WA/14 (H5N2) HPAI, Gf/WA/14 (H5N8) HPAI, and Ws/Mongolia/05(H5N1) HPAI viruses. Each data point represents AI virus titers detected in OP and CL swabs at different days after HPAI virus inoculation. Bars represent thestandard deviations of the mean. All swabs from which virus was not detected were given a numeric value of 101.5 EID50/ml.

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species. Different than the other non-Gs/GD H5 viruses, Tern/South Africa/61 (H5N3) did shed by the cloacal route. This viruscaused a die-off in common terns in South Africa in 1961 and wasnever identified in gallinaceous species, and it therefore remainedadapted to wild bird species (5). As for the nine H7 HPAI viruses,surprisingly, most viruses were shed at similar titers by the OP and

CL route, with CL shedding lagging 2 days, most likely because thei.n. route of inoculation was used to infect the ducks. Viral RNAwas detected in OP and CL swabs for at least 11 dpi and all virusestransmitted to contacts. Based on the pattern of virus shed, mostof these H7 HPAI viruses did not seem particularly gallinaceousadapted, which could be explained if these viruses were isolated

FIG 7 Mean viral shedding in mallards inoculated with Ml/OH/87 (H7N8) LPAI, Ml/Sweden/02 (H7N7) HPAI, Ck/Chile/02 (H7N2) HPAI, and Ck/Canada/05(H7N3) HPAI viruses. Each data point represents AI virus titers detected in OP and CL swabs at days after HPAI virus inoculation. Bars represent the standarddeviations of the mean. All swabs from which virus was not detected were given a numeric value between 101.5 and 101.8 EID50/ml.

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FIG 8 Mean viral shedding in mallards inoculated with Ck/Jalisco/12 (H7N3) HPAI, Ck/Victoris/85 (H7N7) HPAI, Ck/North Korea/05 (H7N7) HPAI,Ck/Netherlands/03 (H7N7) HPAI, and Tk/Italy/99 (H7N1) HPAI viruses. Each data point represents AI virus titers detected in OP and CL swabs at different daysafter HPAI virus inoculation. Bars represent the standard deviations of the mean. All swabs from which virus was not detected were given a numeric value between101.5 and 1.8 EID50/ml.

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early in the outbreaks in poultry or if the outbreaks were short-lived. On the other hand, the pattern of virus shedding of theGs/GD H5 HPAI viruses was what would be expected for viruseswell adapted to gallinaceous species with higher titers in OP swabsthan CL swabs. These viruses were shed at high titers at 2 and 4 dpi,with the highest titers observed with Ws/Mongolia/05 (H5N1),which explains the mortality seen in mallards infected with thisvirus. The Gs/GD H5 HPAI lineage is unique because the virus has

been endemic for 20 years in gallinaceous and domestic duck spe-cies. This complex interplay in multiple species appears to result inthe selection of viruses with characteristics of both gallinaceousand duck-adapted viruses. Viral titers in tissues were also the high-est for these three viruses compared to the other HPAI virusesused in the study, with the exception of Ck/Chile/02 H7N3, Ck/North Korea/05 H7N7, and Tk/Italy/99 H7N1, which also repli-cated to high titers in some tissues.

FIG 9 Mean viral shedding in mallards inoculated with Ck/PA/83 (H5N2) HPAI, Ck/Queretaro/95 (H5N2) HPAI, Tk/Ireland/83 (H5N8) HPAI, and Tern/South Africa/61 (H5N3) HPAI viruses. Each data point represents AI virus titers detected in OP and CL swabs at different days after HPAI virus inoculation. Barsrepresent the standard deviations of the mean. All swabs from which virus was not detected were given a numeric value between 101.5 and 101.8 EID50/ml.

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The Gs/GD H5N1 HPAI viruses developed the unique capacityamong HPAI viruses to replicate well and cause disease in domes-tic and wild ducks, producing a range of syndromes from asymp-tomatic respiratory and digestive tract infections to severe sys-temic infection and death, as seen in gallinaceous poultry (10).Despite efforts to control the spread of H5N1 HPAI viruses, theseviruses continue to evolve, which has led to the emergence ofmultiple genotypes or sublineages (67). The endemic status ofH5N1 HPAI viruses in Asia and Africa has also led to the genera-tion of reassortant H5 strains with novel gene constellations. Re-cently, new subtypes of H5 HPAI viruses (H5N2, H5N5, H5N6,and H5N8) with the genetic backbone of clade 2.3.4 viruses havebeen detected in wild birds, ducks, geese, quail, and chickens (23,29, 32, 67–69). In November and December 2014, HPAI viruses ofthe H5 subtype originating from China were detected in wild birdsand poultry in various countries of Asia and Europe and, for thefirst time, in North America, where the viruses rapidly spread towild waterfowl, wild and captive birds of prey, and backyard andcommercial poultry (22, 30, 34, 70). The involvement of wild birdsin both Europe and North America suggested that these H5 HPAIviruses were better adapted to waterfowl than historic HPAI vi-ruses. This potential adaptation may relate to decreased pathoge-nicity, increased viral shedding, changes in shedding patterns andduration, or a decrease in infectious dose.

Other experimental studies using clade 2.3.4.4 H5N8 HPAIviruses showed that they replicated systemically and were lethal inchickens but appeared to be attenuated, although efficiently trans-mitted, in ducks (71). A range of outcomes of infection—from noclinical signs to severe disease—were observed in ducks i.n. inoc-ulated with H5N8 viruses, and the mortality rates varied from 0 to20% (27, 54, 69, 72, 73). Viral shedding and replication in tissueswas high, and virus was shed for more than 5 days (72). Naturalinfection of domestic ducks with H5N8 has been associated withdrops in egg production and a mild increase in mortality (74), andH5N8 has been detected from carcasses of wild birds, including

mallards (23, 32, 70). However, with these natural infections,other factors could have affected disease presentation, includingcoinfection with other pathogens. In our study, although theducks inoculated with the Gf/WA/14 (H5N8) virus were febrileand the virus replicated well in many tissues, no mortality wasobserved. Ducks challenged with the reassortant H5N2 virus (Np/WA/14) were not febrile and shed virus longer, which would favordissemination and transmission of this virus. This virus exhibitedthe widest geographic spread in the United States. The H5N2HPAI virus is composed of five gene segments (PB2, PA, HA, M,and NS) related to the Eurasian HPAI H5N8, and the remaininggene segments (PB1, NP, and NA) are related to North Americanlineage waterfowl viruses (32, 34, 35). Interestingly, clinical signsand mortality have been described in domestic ducks infectedwith Gs/GD lineage H5 clade 2.3.4.4 HPAI viruses (75). Experi-mental infection of juvenile Muscovy ducks (Cairina moschata)with another reassortant virus belonging to clade 2.3.4.4, A/chicken/BC/FAV-002/2015 (H5N1), caused neurological signs and deathand transmitted to naive contact ducks (75). Muscovy ducks aremore susceptible to HPAI virus infection than other domesticducks, which explains the differences observed when comparingthese results to those obtained in mallards infected with the H5clade 2.3.4.4 viruses in our study (76). The PB1, PA, NA, and NSgene segments of this H5N1 virus were of North American lineage,whereas PB2, HA, NP, and M were derived from the Eurasianlineage H5N8 virus. The role of the genetic makeup of these vi-ruses in the differences observed in pathogenicity remains to bestudied.

The ability of these novel reassortant H5 viruses to replicateefficiently without killing the infected ducks allows them to circu-late within the duck population and increases the possibility oftransmission to poultry. In nature, waterfowl are exposed to manyAI viruses, so some degree of homosubtypic and heterosubtypicimmunity is expected (77). This immunity could afford the duckssome protection; however, if the AI virus is highly infectious, itmight still replicate in the ducks and cause ameliorated clinicalsigns, and normal bird behavior (e.g., migration) may not be im-paired. Based on the viral shedding and transmission results, cou-pled with the lack of clinical signs, many of the H7 HPAI virusesshould be equally fit to transmit efficiently among ducks, yet de-tection of infection in domestic or wild ducks is rare, restricted tothe detection of an H7N1 HPAI virus in domestic ducks duringthe 1999-2000 outbreaks in Italy. This suggests that the lack ofspillover of HPAI viruses from poultry back into wild ducks is notfrom biological incompatibility but from insufficient exposure toinitiate and sustain infection. This underlines the importance ofquickly containing HPAI outbreaks in poultry and the separationof domestic and wild birds.

In summary, in this study, 12 of 14 H5 and H7 HPAI virusesinfected all inoculated and direct-contact mallards, and virus shedwas detected for at least 7 days. The highest titers of virus shedwere detected in ducks infected with the Gs/GD lineage H5 HPAIviruses, but in contrast to shedding patterns observed with LPAIwaterfowl viruses, higher titers were detected in OP swabs than inCL swabs. The comparison of mallards in a standard challengemodel is critical to improve our understanding of virus infectionin ducks, but the information should be carefully extrapolated toall duck species. Previous studies have shown differences in clini-cal disease, shedding, and mortality depending on the duck speciestested (17). In addition, for reasons we do not understand, the

TABLE 4 Comparison of AI virus titers in lungs, spleen, brain, muscle,and hearta

Expt and virus

Virus titer (log10 EID50/g)

Lung Spleen Brain Heart Muscle

Expt 1Ml/MN/00 (H5N2) LPAIV –/– 1.3/– –/1.0 –/– –/–Np/WA/14 (H5N2) HPAIV 6.1/6.8 4.7/6.4 4.1/4.2 4.7/4.6 3.7/5.2Gf/WA/14 (H5N8) HPAIV 5.2/7.2 2.1/4.6 6.1/5.0 3.7/4.3 4.7/5.1Ws/Mongolia/05 (H5N1) HPAIV 7.6/7.4 6.7/6.8 8.0/8.9 8.5/8.0 ND

Expt 2Ml/OH/87 (H7N8) LPAIV 3.4/– 3.1/– –/– ND NDMl/Sweden/02 (H7N7) LPAIV –/3.2 –/3.3 2.1/– ND NDCk/Chile/02 (H7N3) HPAIV 7.3/5.5 5.3/3.4 4.2/3.6 ND NDCk/Canada/05 (H7N3) HPAIV 3.6/4.2 2.7/2.1 –/2.4 ND NDCk/Jalisco/12 (H7N3) HPAIV 3.5/3.7 2.6/4.3 –/5.2 ND NDCk/Victoria/85 (H7N7) HPAIV 2.2/2.1 3.0/1.7 2.1/2.3 ND NDCk/North Korea/05 (H7N7) HPAIV 5.2/5.7 4.6/5.1 2.6/2.8 ND NDCk/Netherlands/03 (H7N7) HPAIV 2.0/2.4 3.0/3.1 2.0/2.0 ND NDTk/Italy/99 (H7N1) HPAIV 6.6/4.9 3.8/4.1 4.0/3.0 ND NDCk/PA/83 (H5N2) HPAIV 1.9/1.7 1.6/1.9 2.1/– ND NDCk/Queretaro/95 (H5N2) HPAIV –/– 2.2/1.8 1.8/1.9 ND NDTk/Ireland/83 (H5N8) HPAIV 2.8/– 3.6/1.2 2.9/2.5 ND NDTern/South Africa/61 (H5N3) HPAIV 3.5/2.4 3.8/3.7 2.3/2.5 ND ND

a Tissues were taken from two ducks per group at 4 dpi (values are expressed in theform “duck 1/duck 2”). –, negative; ND, not done.

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presence of multiple basic amino acids or insertion of amino acidsat the HA cleavage site is predictive of severe disease in gallina-ceous species, but it is not predictive of severe disease in ducks.The high virus titers and the minimal clinical signs observed withthe clade 2.3.4.4 H5 viruses likely represent factors that facilitatetransmission on of these viruses in susceptible wild duck popula-tions. However, transmission and maintenance of avian influenzain wild bird reservoirs is complex, and it remains to be seenwhether these viruses are fit enough to persist in these popula-tions.

ACKNOWLEDGMENTS

The authors appreciate the technical assistance provided by Kira Moresco,Scott Lee, and Nicolai Lee and the animal care provided by Keith Craw-ford, Gerald Damron, and Roger Brock in conducting these studies.

The contents of this study are solely the responsibility of the authorsand do not necessarily represent the official views of the USDA or NIH.Mention of trade names or commercial products in this publication issolely for the purpose of providing specific information and does notimply recommendation or endorsement by the U.S. Department of Agri-culture.

FUNDING INFORMATIONThis research was supported by USDA/ARS CRIS project 6612-32000-063-00D and by the Center for Research on Influenza Pathogenesis(CRIP) and the NIAID-funded Center of Excellence in Influenza Researchand Surveillance (CEIRS; contract HHSN272201400008C).

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