Identification of Type II Interferon Receptors in Geese: Gene ...

Research ArticleIdentification of Type II Interferon Receptors in GeeseGene Structure Phylogenetic Analysis and Expression Patterns

Hao Zhou1 Shun Chen123 Yulin Qi1 Qin Zhou1 Mingshu Wang123 Renyong Jia123

Dekang Zhu23 Mafeng Liu1 Fei Liu3 Xiaoyue Chen23 and Anchun Cheng123

1 Institute of Preventive Veterinary Medicine Sichuan Agricultural University Chengdu Sichuan 611130 China2Avian Disease Research Center College of Veterinary Medicine of Sichuan Agricultural University Chengdu Sichuan 611130 China3Key Laboratory of Animal Disease and Human Health of Sichuan Province Sichuan Agricultural University ChengduSichuan 611130 China

Correspondence should be addressed to Shun Chen sophia cs163com and Anchun Cheng chenganchunvip163com

Received 10 March 2015 Accepted 9 July 2015

Academic Editor Niwat Maneekarn

Copyright copy 2015 Hao Zhou et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Interferon 120574 receptor 1 (IFNGR1) and IFNGR2 are two cell membrane molecules belonging to class II cytokines which playimportant roles in the IFN-mediated antiviral signaling pathway Here goose IFNGR1 and IFNGR2 were cloned and identifiedfor the first time Tissue distribution analysis revealed that relatively high levels of goose IFN120574mRNA transcripts were detected inimmune tissues including the harderian gland cecal tonsil cecum and thymus Relatively high expression levels of both IFNGR1and IFNGR2 were detected in the cecal tonsil which implicated an important role of IFN120574 in the secondary immune system ofgeese No specific correlation between IFN120574 IFNGR1 and IFNGR2 expression levels was observed in the same tissues of healthygeese IFN120574 and its cognate receptors showed different expression profiles although they appeared tomaintain a relatively balancedstate Furthermore the agonist R848 led to the upregulation of goose IFN120574 but did not affect the expression of goose IFNGR1 orIFNGR2 In summary trends in expression of goose IFN120574 and its cognate receptors showed tissue specificity as well as an age-related dependency These findings may help us to better understand the age-related susceptibility to pathogens in birds

1 Introduction

The interferon (IFN) 120574 cytokine can be induced by pathogensor artificial stimulation which subsequently activates antivi-ral antiproliferative and immunomodulatory effects throughrecognizing specific receptors on the surface of target cells[1 2] The IFN120574 receptor (IFNGR) a heterodimer consistingof two chains IFNGR1 and IFNGR2 can be activated by IFN120574to transduce the downstream antiviral signal [3] IFNGR1 andIFNGR2 are single transmembrane (TM) proteins belongingto the class II cytokine family which likely function as thegateway to the control of IFN-mediated cellular signaling Asthe ligand-binding subunit IFNGR1 possesses an intracellu-lar binding site for Janus tyrosine kinase (JAK) 1 a signaltransducer and activator of transcription 1 (STAT1) [1] TheJAK2 binding site is located in an intracellular domain ofIFNGR2 which serves as a signal-transducing subunit [1] All

of these sites are essential for the recruitment and activationof JAK1JAK2 and subsequent phosphorylation of STAT1The activated STAT1 homodimer then translocates to thenucleus and binds to the regulatory sequence (IFN120574-activatedsequence) to promote gene transcription [4 5] MoreoverIFN120574 can also regulate the antiviral gene transcription viaIFN-stimulated gene factor 3 (ISGF3) thus inducing aneffective immune response [6]

Todate studies have focusedmainly onmammalian IFN120574systems but little attention has been paid to avian IFN120574 andits receptors Chicken IFNGR1 was cloned from peripheralblood lymphocytes (PBLs) using the rapid amplification ofcDNA ends (RACE) and the three-dimensional structure ofits extracellular region was identified [7] The extracellularregion of chicken IFNGR2 also shares a similar structure withits human IFNGR counterpart [8] In chickens assessmentof age-related expression of IFN IFN receptors and pattern

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 537637 14 pageshttpdxdoiorg1011552015537637

2 BioMed Research International

recognition receptors (PRRs) has indicated that the IFN sys-tem is somewhat immature during the early developmentalstage of chick embryonic cells [9] The development of IFN120574in the intestinal immunity of juvenile chickens has beencharacterized as well [10]

Based on a comprehensive review of reports on the genestructure evolutionary analysis and crosstalk between IFNand its cognate receptors in birds [11] studies of the IFNsystem in waterfowl appear to be lagging behind In additionthe development and immune characteristics of avian IFN120574are still poorly understood Moreover the duck IFNGR1 andIFNGR2 genes are only predicted sequences Up to now noinformation has been made available on the identification ofgoose IFN receptors Given these considerations this studywas conducted to examine the expression level of goose IFN120574and its associated receptors throughout the embryogenesisphase and posthatch period Herein for the first time gooseIFNGR1 and IFNGR2 cDNA sequences were identified andthe corresponding amino acid sequences as well as structuralcharacteristics were analyzed Comparative analysis of gooseIFNGR sequences with those in birds mammals fish andreptiles may shed light on the evolutionary position of goosegenes among vertebrates The tissue distribution and age-related expression of goose IFN120574 and IFN120574 receptors alsowere analyzed in this study The results of this study willextend existing information on the age-related developmentof goose IFN120574 and its cognate receptors which may shedfurther light on IFN antiviral responses in this species

2 Methods

21 Animals The study was conducted with Sichuan WhiteGeese (Chinese goose A cygnoides) Goose embryos at 20embryonic incubation days (EID20) goslings (1 week of age)and adult geese (3 months of age) were chosen All animalsin this study were purchased from the farm at SichuanAgricultural University (Yarsquoan city Sichuan province) One-week-old goslings and adult geese were maintained for 3days in laboratory animal rooms for acclimation prior toexperiments and water and fodder were provided Thewelfare of the animals was ensured during the samplingprocess

22 RNA Extraction and cDNA Synthesis The birds wereeuthanized and then tissues were collected and snap-frozenin liquid N

2 The chosen tissues included cecal tonsil liver

lung kidney harderian gland brain bursa of Fabriciuscecum heart small intestine spleen thymus gizzard andproventriculus Total RNAwas extracted from various tissuesusing Trizol reagent (Invitrogen Carlsbad CA USA) accord-ing to themanufacturerrsquos instructionsThe cDNAwas synthe-sized using the QuantScript RT kit (Promega Madison WIUSA) according to the manufacturerrsquos instructions FinallycDNA templates of all different samples were stored at minus80∘Cuntil use

23 Molecular Cloning of Goose IFNGR A partial sequenceof goose IFNGR was amplified by the degenerate primersF1 R1 F2 and R2 (all primer sequences used in this

study are listed in Table 1) which were designed based onthe conserved regions among its counterparts in birds (allreference sequences used in this study are listed in Table 2)The resultant PCR fragments were subcloned into the pGEM-T Easy Vector (Promega) followed by transformation ofDH5120572 cells The positive clones were sequenced by using theABI 3730 XL sequencer (Applied Biosystems Foster CityCA USA) Subsequently 31015840 and 51015840 rapid amplification ofcDNA ends (RACE) was performed to obtain the full-lengthcDNA sequence of target genes Based on the partial sequenceobtained Gene Specific Primers (GSPs) including 3GSP13GSP2 5GSP1 5GSP2 and 5GSP3 were designed to obtainthe full-length goose IFNGR cDNA For 31015840-RACE the firststrand cDNAwas synthesized using theAdapter Primer (AP)The 31015840-end of goose IFNGR was amplified by nested PCRusing the primers 3GSP1 and 3GSP2 with AP1 and AP2 For51015840-RACE the first strand cDNAwas synthesized by using theprimer 5GSP1 andM-MLVReverse Transcriptase (Promega)A homopolymeric tail was then added to the 31015840-end of thecDNA using TdT and dCTP (TaKaRa Kyoto Japan) The51015840-end of goose IFNGR was also obtained by two nestedPCRs with the primer pairs 5GSP2Abridged Anchor Primer(AAP) and 5GSP3Abridged Universal Amplification Primer(AUAP) Finally the full-length coding sequence of gooseIFNGR was amplified by using Primer STAR Max DNApolymerase (TaKaRa)

24 BioinformaticAnalysis of Sequences Potential open readingframes (ORFs) were analyzed by using the ORF finderprogram (httpwwwncbinlmnihgovgorfgorfhtml) andtranslated into the corresponding amino acids using DNA-MAN N-Glycosylation sites were predicted with onlinesoftware (httpwwwcbsdtudkservicesNetNGlyc) After-wards the TM region was examined with the TMHMMserver version 20 (httpwwwcbsdtudkservicesTMHMM)The potential protein domains of amino acid sequences wereforecasted via the SMART server (httpsmartembl-heidel-bergde) Pairwise identity analysis was performed withthe Species Demarcation Tool [12] Alignment of putativeamino acid sequences of IFNGR1 and IFNGR2 was per-formed using the Clustal program and sequence similaritieswere calculated with the MegAlign program Secondarystructures were analyzed using the I-TASSER program(httpzhanglabccmbmedumichedu) To analyze the evo-lutionary relationships between type II IFN receptors in birdsand other vertebrates a phylogenetic tree was constructedusing amino acid sequences via the neighbor-joining (NJ)method in MEGA4 with bootstrap analysis based on 1000repetitions [13]

25 Tissue Distribution and Age-Related Expression Analysisof Goose IFNGR mRNA The tissue distribution of IFNGRin healthy 1-week-old goslings was studied by real-timequantitative qPCR (RT-qPCR) using the Bio-Rad CFX96Real-Time Detection System The age-related expressionanalysis of goose IFN120574 receptors at the mRNA level incertain tissues of geese (embryonic incubation 20 days andadult) was also detected by RT-qPCR Where possible theprimers were designed across intron and extron boundaries

BioMed Research International 3

Table 1 List of primers and sequences

Methods Gene name Primer name Nucleotide sequence (51015840-31015840)Reverse transcription Oligo(dT)18 TTTTTTTTTTTTTTTTTT

Partial sequenceIFNGR1 F1 TTAAAGCTGTTGTTGGATCA

R1 CAATCACASGYTGTTCTTC

IFNGR2 F2 CTGAGGTGGTCTCCTGTTAR2 TCAAATACTCTTCAAWGTGTG

3RACEAP CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC (T)18AP1 CCAGTGAGCAGAGTGACGAP2 GAGGACTCGAGCTCAAGC

5RACE AAP GGCCACGCGTCGACTACGGGIIGGGIIGGGIIGGGIIGAUAP GGCCACGCGTCGACTAGTAC

3RACE-GSPIFNGR1 G1-3GSP1 GGCACCAGACAAAGTGGAAGAGTC

G1-3GSP2 TGCAGAAGATTACAGAGGAGGTCC

IFNGR2 G2-3GSP1 TGGACTGCGGAGAATCCCGGAATGG2-3GSP2 CAATGAGTGAGACAACCAGAGCTG

5RACE-GSP

IFNGR1G1-5GSP1 ATCCCAAAAAGTCACCG1-5GSP2 GAAATACAGGATGGTAAATATCAACG1-5GSP3 GAGAGATCCAGTTTTGGAGGTC

IFNGR2G2-5GSP1 CATTCTCCCAGTAGG2-5GSP2 AGTCACGCTGTTCACTTTAGGGG2-5GSP3 ATTCCACCCAGTCAGAAGTCAT

Real-time PCR

IFNGR1 goqRT-G1-F GCATTCAGGTTCCTCTTGgoqRT-G1-R AAGCGTTATCCATGTTCAG

IFNGR2 goqRT-G2-F AATCTTCTCCACGTTTACCGgoqRT-G2-R CAGTAGAAGTAATTCATGGTG

120573-actin goqRT-120573actin-F TCCCTGGAGAAGAGCTACGAgoqRT-120573actin-R GTGTTGGCGTACAGGTCCTT

Degenerate bases Y = C + T W = A + T and S = C + G

Table 2 List of reference sequences

Gene name Organism GenBank accessionnumber

IFNGR1

Ficedula albicollis XM005043892Taeniopygia guttata XM002194727

Gallus gallus NM001130387Anas platyrhynchos XM005017754

IFNGR2


Gallus gallus AY820753Anas platyrhynchos XM005013846

Reactionswere carried out in triplicate each in a total reactionvolume of 10 120583L including 08120583L cDNA sample 5 120583L SYBRGreen PCR master mix (QuantiFast SYBR Green PCR Kit)03 120583L of each primer (listed in Table 1) and 36 120583L ddH

2O

The amplification program was 94∘C for 4min followedby 40 cycles of 94∘C for 10 s and 58∘C for 30 s After theamplification phase a melting curve analysis (from 65∘C to95∘C with a heating rate of 05∘C per second and a continu-ous fluorescence measurement) was routinely performed to

confirm the presence of a single and specific PCR productStandard curves were generated for each gene from 10-foldserial dilutions of PCR products to estimate amplificationefficiency Finally RT-qPCRdatawere analyzed by the 2minusΔΔCTmethod using Bio-Rad CFX Manager Software

26 Transcriptional Levels of IFN120574 and IFNGR in GooseMononuclear Cells (MNCs) after R848 Stimulation Goose(3 months of age) spleen MNCs were collected culturedin RPMI1640 (Gibco Gaithersburg MD USA) and thenseeded into 24-well cell culture plates in 10 serum-containing RPMI1640 medium Thereafter the cells werestimulated with R848 (20120583gmL) (Invivogen San DiegoCA USA) for 10 h while PBS-treated cells were chosen as acontrol IFN120574 and IFNGR transcripts were detected by RT-qPCR according to methods described above

3 Results

31 Sequence Analysis of Goose IFNGR1 The full-length(1322 bp) cDNA of goose IFNGR1 [GenBank KM457284]contains a 117 bp 51015840-UTR a 1134 bp single open reading frameencoding 377 amino acids and a 71 bp 31015840-UTR (Figure 1)Three potential N-glycosylation sites were found in the


aagcagccgcccgcagtgccttcaccaacaaaccttgtagtaacatcccaaaatttcaaaaccgtcttgagttggcagtaccagcctacg

tctgaaactccttattttgttgtggaaATGAAACCTTACAGCCCAGGTACCTATATGACTGTTTCAACTTGTGTGAACATCTCAACTAAT

M K P Y S P G T Y M T V S T C V N I S T N

TCTTGTGATCTCTCACGGGAAGTAAAGGAAACTTTTTCTCCTTACTGGTTTCGAGTTAAAGCTGTTGTTGGATCAGAACAGTCTGAGTAT

S C D L S R E V K E T F S P Y W F R V K A V V G S E Q S E Y

GTTGAAACAAATGAGTTTATTTTGCAAAAGCATGGAAAAATAGGACCTCCAAAACTGGATCTCTCAAGGCATGCTGATAAAATCATAGTT

V E T N E F I L Q K H G K I G P P K L D L S R H A D K I I V

GATATTTACCATCCTGTATTTCCATCTATGGAGTTTCAGCCTTGGATCACAGACAATTTAGATTTCATGTACAAGGTGACTTTTTGGGAT

D I Y H P V F P S M E F Q P W I T D N L D F M Y K V T F W D

AATGAAACTCAGCGTAAAGAAGAGGTTTTTGCAGAAGACTGTCAGGTGGATAAATGTAGCCTAGACATCCCAGTTACTCCTAATGGTTCT

N E T Q R K E E V F A E D C Q V D K C S L D I P V T P N G S

ATTTACTGTGTTTCGGCAAAGAGCAGTTTGTTTGAAAATCTGATAGTTGGTGCCCCGTCCGAAGAAAGCTGCATTCAGGTTCCTCTTGAG

I Y C V S A K S S L F E N L I V G A P S E E S C I Q V P L E

CAAACTACGAGTACACAAAACATTGTCATTGTGTGCGTGGCTGTGGTGATCATGGGCATCATTTTGACATTATGTTGTGGCTTCAAGAAG

Q T T S T Q N I V I V C V A V V I M G I I L T L C C G F K K

CTAAGGGAGAGGAATATAAAGCTGCCTAAATCCTTGGTCACTGTGATAAGAAACCTGAACATGGATAACGCTTTAGAATCAAAATCAGAG

L R E R N I K L P K S L V T V I R N L N M D N A L E S K S E

GGAAAATACATCTCTATAGTAAGCGTCATGCCAGTCCAGTCAGCGTTGCCTTTGAATAGCAAAGAAGCCTTGCTGAATATAGAGCCAGAA

G K Y I S I V S V M P V Q S A L P L N S K E A L L N I E P E

GAAGAAGCTGTCAGTCTTGATAATTTCAGTGAAGGAGCATCTTCTTGTCCTCCGCCAGAGGCACCAGACAAAGTGGAAGAGTCCTCTGTG

E E A V S L D N F S E G A S S C P P P E A P D K V E E S S V

CAGAAGATTACAGAGGAGGTCCCTTCTGATGATGAACAGAATTGTAAAGTAAAAGAGAGTTACTTTATTTCGGACAGTAACCAAACAGGT

Q K I T E E V P S D D E Q N C K V K E S Y F I S D S N Q T G

ATAAGTAGTAACTCTTCAGGTCCAGAGGTTTCTGCCACAGAAATACAACAAACAGTCATTCCAAGAAGCTGTCCCAAATTTTCTGGCTAT

I S S N S S G P E V S A T E I Q Q T V I P R S C P K F S G Y

GACAAGCCCCACGTGCCATTAGATATGTTGATAGATGTTGGTGAAGAACAACCTGTGATTGCTTACAGGCCTACTGACTAAccaggatag

D K P H V P L D M L I D V G E E Q P V I A Y R P T D

atgaaatgtttaataaaagctcatgaagaacagcaaaaaaaaaaaaaaaaaaaaaaaaaaaa

1911181222715236182451112541142631172721202811232901262991292108132211713521261

(a)aggtcggtgccgggcttcgggcgaagaatcttctccacgtttaccggcaccaaaggatgtaaaggtttattcctataacttccacagcgc

gctgaggtggtctcctgttaaagtagatagaggcgtggtgttatatacagtccattttaaaacaggggcctttaaccagtgggatgagat

aaactgcactcgtatcgcccggactgagtgcagtttccccctgtcacttaatgagcgtctctggacttttgttttgcgtgtgaggtctga

gctggggcaaATGACTTCTGACTGGGTGGAATCGGATCCATTTGTGGCAGAGAGAGACACTACCATAGGGCCCCCTAAAGTGAACAGCGT

M T S D W V E S D P F V A E R D T T I G P P K V N S V

GACTGTAAGCTCTGACTCACTGCTCATTAGTGTCTCACCCCCTTTTGAATTCGAAGAAGGTACTCTCCAGTATCATGTGTCCTACTGGGA

T V S S D S L L I S V S P P F E F E E G T L Q Y H V S Y W E

GAATGCAACGACTACTACTAAAGAGATGTTGGTGAATAATGCACTATTCAAAATTGAAAATCTAAAGCAAATGACACTTTATTGTTTTAC

N A T T T T K E M L V N N A L F K I E N L K Q M T L Y C F T

AATTGAAATAGAACTGAAAATGCATTTATATGACCGGATCCCTGGACTGCAGAGAATCCCGGAATGTTACAGAACTCCAATGAGTGAGAC

I E I E L K M H L Y D R I P G L Q R I P E C Y R T P M S E T

AACCAGAGCTGCATATATTATAACAACATTTACACTGGTCGGTCTTGTTTTAATTCTCATAATAATTGGTTTGTTTTGTCTGTGGAGACA

T R A A Y I I T T F T L V G L V L I L I I I G L F C L W R H

TCACAAAACAATTAAGTATTTGTGTCAGCCACCTTTAAAAATCCCATCACACATTGAAGAGTATTTGAGAGACCCTGGCATGCCTCATTT

H K T I K Y L C Q P P L K I P S H I E E Y L R D P G M P H L

AGAAGCGTTGGAGAATTACCACGAGGAAGCTCCACACGATTCTTTATCTGTTTTGTGTTTTGAAGAAGGAAGCGAAGCGTATGATGACAC

E A L E N Y H E E A P H D S L S V L C F E E G S E A Y D D T

TTTGGATGGTAACACTCGTTCACACAGCAGCTCCGGTGACTGTGAAGTAACTTAAgcagtgccccagtgagaatgcctgtttccagtcgt

L D G N T R S H S S S G D C E V T

tgcagaggctcctgcgagtggtgctgtgcagctccatgcaggacagacaaggagccctaaacaacacgtggccattggcaaagcctgagc

aagactttgcttcctgagaataatatggaacctgagcctttttaaaaatattttctgctgtgttacgacaaccttctttctgccaaagag

actgaaaacgtggaagctgaagggaagtgaacactaaattgctttcagacagatggttatggctgtgtcatcattccccccgcccccttg

aagcaaagtactgagaaataatggcactttagggtagggcctagtataacctggaatgtattagggagagagtaattgcaccatgaatta

cttctactggggtttttgttttgttgtcttatttgtgaagcttgtgttgccacttaaagtgtattatatccaaaaaaaaaaaaaaaaa

1911812711361284515854188631118721148811

901178

991208

10811171

13511261

(b)

1 377199177

TM

(c)

1

TM

22414597 12319

FN3

(d)

Figure 1 Nucleotide sequences of goose IFNGRs and deduced amino acid sequence structure (a) Nucleotide sequence of goose IFNGR1and the deduced amino acid sequence The 51015840-UTR and 31015840-UTR sequences are shown in lowercase letters while the ORF is presented inuppercase letters The putative amino acid sequence is highlighted in blue and presented below the capital letters Potential N-glycosylationsites are boxed (b) Nucleotide sequence of goose IFNGR2 and deduced amino acid sequence (c) Predicted protein domains characteristicof IFNGR1 and their alignment with counterparts from other birds and mammals Conserved sequences are represented by the graph underthe alignment TM domains are marked in light yellow (d) Predicted protein domains characteristic of IFNGR2 TM domains are marked inlight yellow while the fibronectin type III domain (FN3) is marked in light red


100279 100315 875 10031 632 595 100

307 598 587 588 100333 584 594 576 897 100318 602 557 54 806 842 100325 58 574 536 784 814 799 100292 591 572 552 799 78 763 776 100308 627 602 586 609 628 593 611 61314 666 635 61 713 696 685 678 706312 616 563 575 596 597 566 588 607313 609 558 563 594 593 565 583 608343 457 449 458 483 461 467 442 484323 455 454 458 45 451 453 444 42529 404 39 418 429 436 433 431 436

269 304 319 281 289 311 318 293 31230 296 319 295 306 318 329 314 286

316 321 338 352 327 335 346 313 351321 323 338 357 327 333 344 313 351326 323 322 344 325 335 348 329 354313 349 359 353 344 358 378 364 35274 341 33 325 304 315 332 34 32306 322 323 335 319 321 337 298 325309 333 337 337 309 352 34 316 31528 314 325 348 363 344 368 306 34

321 352 325 285 289 295 302 289 274195 257 253 257 224 224 229 222 226235 219 24 228 228 254 235 231 215

100683 100631 729 100627 715 964 100456 487 457 45 100441 474 455 447 723 100427 467 441 447 508 504 100301 292 305 307 319 301 311 100296 301 298 294 36 317 343 74 100317 33 336 345 374 353 378 502 533 100315 33 336 345 374 35 376 502 538 994 100308 323 333 343 371 348 38 511 535 953 951 100348 365 359 356 408 383 384 492 474 616 621 623 100299 329 325 335 36 355 345 495 492 607 611 603 755 100319 319 311 315 365 331 373 496 487 609 613 612 726 73 100316 34 333 345 359 352 363 509 483 591 596 597 715 702 951 100332 339 322 32 377 346 328 291 308 325 323 337 317 308 284 288323 311 30 301 327 313 286 282 283 319 31 311 299 285 282 276229 248 245 234 251 264 217 211 226 21 21 202 231 232 215 225248 207 233 241 233 239 256 225 241 245 241 239 222 238 236 23

100401 100193 204 100237 205 258 100

gtAAI67921_Xenopus_Silurana_tropicalisgtgoose_IFNGR1gtXP_005017811_Anas_platyrhynchosgtNP_001123859_Gallus_gallusgtXP_005489284_Zonotrichia_albicollisgtXP_005420190_Geospiza_fortisgtXP_002194763_Taeniopygia_guttatagtXP_005043949_Ficedula_albicollisgtXP_005518707_Pseudopodoces_humilisgtXP_005509491_Columba_liviagtXP_005154885_Melopsittacus_undulatusgtXP_005435859_Falco_cherruggtXP_005230295_Falco_peregrinusgtXP_005280397_Chrysemys_picta_belliigtXP_006112786_Pelodiscus_sinensisgtXP_006030325_Alligator_sinensisgtEDL93784_Rattus_norvegicusgtEDL03452_Mus_musculusgtXP_003898168_Papio_anubisgtNP_001253229_Macaca_mulattagtAAH05333_Homo_sapiensgtXP_006188549_Camelus_ferusgtBAN09008_Sus_scrofagtAAI03297_Bos_taurusgtXP_004011420_Ovis_ariesgtAGU16999_Anolis_carolinensisgtETE71008_Ophiophagus_hannahgtAAI63407_Danio_reriogtNP_001117888_Oncorhynchus_mykiss

(a)

100471 100302 374 100308 347 797 100346 34 748 747 100282 272 578 602 525 100326 368 488 498 509 408 100342 40 477 512 578 41 711 100363 387 51 521 521 413 721 748 100369 39 51 528 521 417 723 752 91329 353 464 441 498 448 578 603 622

315 364 435 459 459 356 59 632 627352 382 50 483 511 421 629 657 656307 343 317 324 308 246 328 377 355306 353 366 349 34 284 344 362 36533 361 335 308 329 254 335 37 343

317 36 335 312 312 254 332 366 34326 365 339 315 319 25 338 374 34234 367 347 309 321 262 318 379 362

303 31 303 302 286 235 333 351 357306 355 327 315 311 256 339 356 36931 35 291 316 298 261 29 34 324

268 285 268 277 286 262 279 288 285

100622 100614 844 100661 677 61 100361 338 345 337 100369 368 356 338 815 100344 301 34 352 605 599 100352 311 341 353 606 594 958 100342 311 349 354 614 605 952 988 100352 301 327 35 604 591 862 863 865 100353 294 337 333 518 504 637 644 651 626 100366 295 34 314 523 519 636 645 652 635 931 100335 317 333 332 548 548 651 651 655 64 772 755 100297 255 295 314 272 278 27 276 284 286 254 262 285 100

gtAGL76447_Anolis_carolinensisgtJAA96806_Crotalus_horridusgtXP_002189244_Taeniopygia_guttatagtXP_005427852_Geospiza_fortisgtXP_005526690_Pseudopodoces_humilisgtXP_005497041_Zonotrichia_albicollisgtXP_005151848_Melopsittacus_undulatusgtXP_005511438_Columba_liviagtXP_005438664_Falco_cherruggtXP_005234445_Falco_peregrinusgtgoose_IFNGR2gtXP_005013903_Anas_platyrhynchosgtAAV67776_Gallus_gallusgtNP_0011017831_Rattus_norvegicusgtAAC52938_Mus_musculusgtABW97193_Papio_anubisgtJAA36651_Pan_troglodytesgtNP_005525_Homo_sapiensgtACA51056_Callicebus_molochgtAAS59772_Bos_taurusgtXP_004002812_Ovis_ariesgtNP_001104728_Sus_scrofagtABY87189_Oncorhynchus_mykiss

(b)

Figure 2 Heat map of IFNGR sequences in different species The 2D color-coded matrix decorated with a full color spectrum scheme ofIFNGR1 (a) and IFNGR2 (b) based on pairwise identity scores was constructed using the Species Demarcation Tool (STD)

goose IFNGR1 amino acid sequence (Figure 1) Only one TMdomain was identified in goose IFNGR1 indicating that it isa single membrane protein (Figure 1)

Additionally the deduced amino acid sequence of gooseIFNGR1 was compared with those of avian and mammalianspecies According to the 2D color-coded matrix generatedbased on a pairwise sequence alignment analysis (Figure 2)goose IFNGR1 shared the highest identity with its counter-part in Anas platyrhynchos [GenBank XP005017811] (875)which is much higher than that of Homo sapiens [GenBankAAH05333] (323) and Danio rerio [GenBank AAI63407](257) Notably the IFNGR1 amino acid sequence of Gallusgallus [GenBank NP001123859] showed a lower identity withthat of goose (632) than that of duck (875)

The multiple sequence alignment analysis showed thatfive cysteine sites and five tyrosine sites are completelyconserved in birds and mammals (Figure 3) Furthermorethe JAK1 binding site (LPKSLV) and STAT1 binding site(YDKPH) were found in goose IFNGR1 which is highlysimilar to those of human and mouse (Figure 3)

32 Sequence Analysis of Goose IFNGR2 In this study gooseIFNGR2 was also cloned for the first time The full-lengthcDNA of goose IFNGR2 [GenBank KM461716] obtainedwas 1438 bp with an open reading frame of 675 bp encodingfor 224 amino acids (Figure 1) The 51015840-UTR and 31015840-UTRof IFNGR2 were 280 bp and 483 bp in length respectivelyIFNGR2 was predicted to have only one N-glycosylation siteat the 58th amino acid (Figure 1) Unlike goose IFNGR1goose IFNGR2 was found to have a TM domain and afibronectin type III domain (FN3)

The color-coded matrix based on amino acid sequencealignment (Figure 2) showed that goose IFNGR2 shared thehighest identity with A platyrhynchos IFNGR2 [GenBankXP005013903] (844) Meanwhile it shared 677 identitywith G gallus IFNGR2 [GenBank AAV67776] 622 iden-tity with Falco cherrug IFNGR2 [GenBank XP005438664]and 603 identity with Columba livia IFNGR2 [GenBankXP005511438]

The multiple sequence alignment analysis of IFNGR2showed that two cysteine sites and four tyrosine sites were


--------------------------------------------------

-MQADVLAYSGKMREWHYRRSVRPGFLLGGTTKQVKASACRPPTDVTQKA

------------------------------------MGAPLALMVLTALV

MGSTRHLPSRALYRNDPGPSAAGGARPRVATAARCGRREPILEDKVCGQG

--------------------------------------MALLFLLPLVMQ

-----------------------------MGPQAAAGRMILLVVLMLSAK

GooseAnas platyrhynchosGallus gallusTaeniopygia guttataHomo sapiensMus musculus

--------------------------------------------------

AKQAAGAGFSSTLQP-VPSPTDLVVTSQNFKTVLSWQYQPMSETPYFVVE

APGQNAASLQERLPA-VPSPTGTSVKSKNFRTVLYWQYPSMSETPHFVVE

EEDLEAVVVVQVLAAHLPSPTGIVVTSENFKTVLHWQYPTMSKTPHFIVE

GVSRAEMGTADLGPSSVPTPTNVTIESYNMNPIVYWEYQIMPQVPVFTVE

VGSGALTSTEDPEPPSVPVPTNVLIKSYNLNPVVCWEYQNMSQTPIFTVQ


MKPYSPGTYMTVSTCVNISTNSCDLSREVKETFSPYWFRVKAVVGSEQSE

IKPYIPGTYMTVSTCVNISTNSCDLSREVKETFSPYWFRVKAVVGSEESE

VKPYLSGKYQTVSTCVNISATSCDLSEEINEIFHSYWFRIKAIVGSQQSQ

IKPYNLGHYKNVSTCVNTSAHFCDLSKEICDPYSSHWLRVKAVVGSQESE

VKNYGVKNSEWIDACINISHHYCNISDHVGDPSNSLWVRVKARVGQKESA

VKVY---SGSWTDSCTNISDHCCNIYEQIMYPDVSAWARVKAKVGQKESD


YVETNEFILQKHGKIGPPKLDLS-RHADKIIVDIYHPVFPS------MEF

YVETNEFILQKHGKIGPPKLDLS-RHADKIIVDIYHPVFP-------MEL

YVETDEFVLQKHGKIGPPKLNLS-RHGAEIIVDVYHPEFPS------VEV

YVEANEFILQRHGKIGPPKLNIS-RHGDKIMVDIYHPVFP----------

YAKSEEFAVCRDGKIGPPKLDIR-KEEKQIMIDIFHPSVFVNGDEQEVDY

YARSKEFLMCLKGKVGPPGLEIRRKKEEQLSVLVFHPEVVVNGESQGTMF


QPWITDN-LDFMYKVTFWDNETQRKEEVFAEDCQ--VDKCSLDIPVTPNG

QPWITDN-SDITYQVTFWDNETQHKNEVFADDCLQFTNKCSIDIPVTPNG

RPWMREIYSELSYSVIFRNSENESRKNFTVADCE--MNECNLSIPVPSEG

LSCIEDIYSNLAYLVTVQGSENE-TEELYEDNCT--VHKCSLKIPVLTES

DPETTCYIRVYNVYVRMNGS-EIQYKILTQKEDDCDEIQCQLAIPVSSLN

GDGSTCYTFDYTVYVEHNRSGEILHTKHTVEKEECNETLCELNISVSTLD


SIYCVSAKSSLFENLIVGAPSEESCIQVPLEQTTSTQNIVIVCVAVVIMG

STYCVSAKGILFQNLIVGAPSEESCIQVPLEQTTSTEKMVIVCVAVVIMG

STYCVSAKGHFFDDLIVGASSEESCIWVPITQAWSTQVTIAVSSIVLVVS

STYCVSAKG-IFDSLMVGTPSEESCTPAPLRQTSSTHGIIILCVVIGILT

SQYCVSAEGVLHVWGVTTEKSKEVCITIFNSSIKGSLWIPVVAALLLFLV

SRYCISVDGISSFWQVRTEKSKDVCIPPFHDDRKDSIWILVVAPLTVFTV


IILTLCCGFKKL----RERNIKLPKSLVTVIRNLNMDNALESKSE-----

VIFTLFCGFKKL----REKNIKLPKSLVTVIRNLNTDNTFESKSE-----

LILTVCYGCKKL----RKKNIKLPKSLVSVIRSLNADNSFESRSE-----

VLLTVYCGCKKL----RKNNIQLPKSLVSVMRNLNTGALMGPRSE-----

LSLVFICFYIKKINPLKEKSIILPKSLISVVRSATLETKPESKYVSLITS

VILVFAYWYTKK-NSFKRKSIMLPKSLLSVVKSATLETKPESKYS-LVTP


-------GKYISIVSVMPVQSALP---LNSKEALLNIEPEEEAVSLDNFS

-------GKYISVVSIMPVQSVSP---LNSKETLLNIEPEEEAVSPENFS

-------AKGICAASVMPVPSVSVPLTVNDDEALLNVES-AEDVSPEDFS

-------GKYISVTSRLSDLPVIG------EVTLLEIEPKEQTVSPVNSC

YQPFSLEKE-VVCEEPLSPATVPGMHTEDNPGKVEHTEELSSITEVVTTE

HQPAVLESETVICEEPLSTVTAP-----DSPEAAEQ-EELSKETKALEAG


EGASSCPP--PEAPDKVEESSVQKITEEVPSD-DEQNCKVKESY------

EEASSCPL--PETPDKVEESSVQKITEEVPSD-DEQNCKVKESY------

EGTSSGPP--LEASHKLEETSVQEN-TEVPSD-VEQSHKEKESD------


DGESSVPS--PEAPAKVEEVPIQESTEEVSVDTDEQNCEVKESY------

ENIPDVVPGSHLTPIERESSSPLSSNQSEPGSIALNSYHSRNCSESDHSR

GSTSAMTPDSPPTPTQRRSFSLLSSNQS--GPCSLTAYHSRNGS------

(a)

Figure 3 Continued




---------FISDSNQTGISSNSSGPEVSATEIQQTVIPRSCPKFSGYDK

---------FISSSNQTDTSSNSSGPEISATEIHQTVMPRSCPKFSGYDK

---------FISDSSQTDVCSNSSGPVVSATEIRQAVIPSSCPKFSGYDK

---------FISNSSQVDICSKSSESEISTTETQSTVTPSRCFKFSGYDK

NGFDTDSSCLESHSSLSDSEFPPNNKGEIKTEGQELITVIKAPTSFGYDK

-----DSGLVGSGSSISDLESLPNNNSETKMAEHDPPPVRKAPMASGYDK

PHVPLDML-IDVGEEQPVIAYRPTD------

PHVPIDML-IDVGEEQRVIAYRPTD------

PHVPLDVL-IDVGEEQPVIAYRSTE------

PHVPLDVLMIDVGEEQPVNAYRPTE------

PHVLVDLL-VDDSGKESLIGYRPTEDSKEFS

PHMLVDVL-VDVGGKESLMGYRLTGEAQELS

(b)

Figure 3 Multiple alignment analysis of IFNGR1 amino acid sequences from geese birds and mammalians Selected species and GenBankaccession numbers are as follows A platyrhynchos [XP005017811] G gallus [NP001123859] Taeniopygia guttata [XP002194763] H sapiens[AAH053331] andM musculus [EDL034521] The alignment was generated with ClustalW and modified manually Amino acids conservedamong all species are indicated as identical (lowast) highly conserved () or weakly conserved () The light green shade highlights the JAK1binding site while the light red shade indicates the STAT1 binding site

completely conserved in birds and mammals (Figure 4)Consistent with the human and mouse counterparts gooseIFNGR2 also had a JAK2 binding site (PLKIPSHIEEYL)located in a span from position of 158 to 169 (Figure 4)

33 Secondary Structural Model of Goose IFNGR1 andIFNGR2 As depicted in Figure 5 the secondary structure ofgoose IFNGR1 protein was predicted to contain 3 120572-helicesand 17 120573-sheets Meanwhile the goose IFNGR2 amino acidsequencewas predicted to contain 2120572-helices and 12120573-sheetsAlthough the IFNGR1 amino acid sequence was longer thanthat of IFNGR2 their secondary structures were observed tobe similar

34 Phylogenetic Analysis of Goose IFNGR To clarify theevolutionary relationship between IFNGR of geese and otherspecies a phylogenetic tree was constructed with the aminoacid sequences based on a Poisson model as shown inFigure 6 These sequences were mainly separated into fourclusters of avian mammalian fish and amphibianreptiliangroups The phylogenetic analysis showed that the IFNGR1and IFNGR2 clusters were divergent subgroups Further-more goose IFNGR1 appeared to be closely related to itscounterparts among birds especially duck IFNGR1 Analysisof the bird group also revealed that the goose IFNGR1and duck IFNGR1 sequences were located in the samemonophyletic group which was distinct from other birdssuch as chickens pigeons and sparrows Similar results alsowere observed with goose IFNGR2 Furthermore the geneticdistance of fish sequences analyzed was relatively far fromthose of avian species and goose IFNGR1 and IFNGR2showed the farthest distance from the fish IFNGRmolecules

35 Tissue Distribution of Goose IFN120574 and IFNGR Thequantitative analysis showed that the relative expression levels

of IFN120574 IFNGR1 and IFNGR2 mRNA varied in differenttested tissues (Figure 7) Relatively high levels of IFN120574 weredetected in the harderian gland cecal tonsil and cecumfollowed by thymus liver bursa of Fabricius and spleenand the IFN120574 expression was lowest in the brain Thegoose IFNGR1 gene was highly expressed in the cecal tonsilmoderately expressed in the lung bursa of Fabricius heartand proventriculus andminimally expressed in the brain andgizzard In addition goose IFNGR2 was strongly detectedin the immune-associated tissues especially in the cecaltonsil and bursa of Fabricius Inmost immune-related tissuesthe relative mRNA transcriptional levels of IFN120574 IFNGR1and IFNGR2 were similar at the same time point and theubiquitous expression of these genes in immune tissues ofhealthy goslings was observed

36 Age-Related Expression Analysis of Goose IFN120574 andIFNGR To understand the expression patterns of IFN120574 andits receptors their mRNA levels in ten tissues of gooseembryos goslings and adult geesewere assessed byRT-qPCR(Figure 8) In goose embryos the highest level of IFN120574 wasfound in the cecum while it was barely expressed in thebrain Meanwhile IFNGR1 was detected at high levels in thececum small intestine and liver and at lower levels in theheart kidney harderian gland and bursa of Fabricius In theembryonic stage goose IFNGR2 was strongly transcribed inthe harderian gland and small intestine In the adult gooseIFN120574 was strongly detected in the kidney and harderianglandThe highest level of IFNGR1 was seen in the liver whileIFNGR2 was strongly transcribed in the liver and spleenHowever no significant differences were observed in theexpression of IFNGR2 in the heart lung and thymus

Obvious decreases in IFN120574 expression were observed inthe cecum small intestine and lung during goose develop-ment Notably in the cecum heart harderian gland kidney


Goose --------------------------------------------------MAIFELWTCRPNVPCGVVYLNVLIIIFISFHVLDSSPCLPAPKDVKVYSY---------MPWRPLLLFLVGIFLLGPARAPGTEASPHLPAPEDVMVYSF-----------------------------------------------------------MRPTLLWSLLLLLGVFAAAAAAPPDPLSQLPAPQHPKIRLY

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus ---------MRPLPLWLPSLLLCGLGAAASSP-DSFSQLAAPLNPRLHLY

Goose --------------------------------------------------NFHNTLRWSP--VKVERGVVLYTVHFKTGAFNQWD------EMNCTRIARNFCNSLRWSP--VKVDGGSVSYTVQFKTGAFNHWS------EMDCTRITQ------------------------------------------MGCAQTPRNAEQVLSWEPVALSNSTRPVVYQVQFKYTDSKWFTADIMSIGVNCTQITA

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus NDEQILTWEPSPSSNDPRPVVYQVEYSFIDGSWHR----LLEPNCTDITE

Goose ------------------------------MTSDWVESDPFVAERDTTIGTECSFP----LSLNERLWTFILRVRSELGQMTSDWVETDPFVAERDTTIGTECSFL----KSVKERRWTVVLRVRAEMGPRTSAWVETDPFVAERNTTIGTWCPFP----PELRRRRWTILLRLRAERGALASPWVLTPPFVAETNTTLGTECDFTAASPSAGFPMDFNVTLRLRAELGALHSAWVTMPWFQHYRNVTVG

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus TKCDLTGGGRLKLFPHPFTVFLRVRAKRGNLTSKWVGLEPFQHYENVTVG

Goose PPKVNSVTVSSDSLLISVSPPFEFEEG---TLQYHVSYWENA-TTTTK-EPPKVNSVIVSSDSLLISVSPPFESKEG---TVQYKVSYWENA-TTATKEEPPKVNSVIVSSDSLLISVTPPFGPEPGY--HLQYHVSYWENT-TITTKKEPPRVNNVSARPDSLLVGVSPPFTPEPGD--LLQYLVSYWENS-SSPTEKKPPENIEVTPGEGSLIIRFSSPFDIADTSTAFFCYYVHYWEKGGIQQ--VK

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus PPKNISVTPGKGSLVIHFSPPFDVFHG--ATFQYLVHYWEKSETQQEQVE

Goose MLVNNALFKIENLKQMTLYCFTIEIELKMHLYDRIP-GLQRIPECYRTPMMWVNNALFKIENLKQMTLYCFTIEIELVKYLHEQIP-GLQRIPECYRTPMIKTSNTLFKIKDLKQSTLYCFTIQIELMTYSRFHLI-GLQTVPECYRTTILSESKTRFEIGNLKESTLYCFSIQVQLKIYSGHLLE-GQQSAPECHRTALGPFRSNSISLDNLKPSRVYCLQVQAQLLWNKSNIFRVGHLSNISCYETMA

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus GPFKSNSIVLGNLKPYRVYCLQTEAQLILKNKKIRPHGLLSNVSCHETTA

Goose SETTRAAYIITTFTLVGLVLILIIIGLFCLWRHHK-TIKYLCQPPLKIPSNETTRVVYIITTFTLVGLVLILMIIGLFFLSRHHK-TIKYLCQPPLKIPSSEATKAGYIVAIFMSVGLLLIVIIVGFFCLWRNQK-AIKYLSQPPLRIPSSEATRAWYIIFLFSVGFVALNLVVAASLFLWKYHQ-KIKYWAQPPLEIPSDASTELQQVILISVGTFSLLSVLAGACFFLVLKYRGLIKYWFHTPPSIPL

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus NASARLQQVILIPLGIFALLLGLTGACFTLFLKYQSRVKYWFQAPPNIPE

Goose HIEEYLRDPGMPHLEALENYHEEAPHDSLSVLCFEEGSEAYDDTLDGNTRHIEEYLRDPSMPHLEALENHPEEALPDSYSVLYFEEGSKAYGDTLAEDTRHFEEYLRDPSMPQLEVLENHDED-PQDLLTVVYTGEGSSAYGDMLDGNTCHFREFLRDPDVAGLEELYSPAEE---EPQALVLGGEGGQEGEDPSPNTSRQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPEKEQEDVLQTL--

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus QIEEYLKDPDQFILEVLDKDGSPKEDSWDSVSIISSPEKERDDVLQTP--

Goose SHSSSGDCEVTSHSSSSESEVTSHSSSSSRDVTARAASEGPPQ------------

Anas platyrhynchos Gallus gallus Taeniopygia guttata Homo sapiensMus musculus -----------

Figure 4 Multiple alignment analysis of IFNGR2 amino acid sequences from several birds and mammals Selected species and GenBankaccession numbers are as follows A platyrhynchos [XP005013903] G gallus [AAV67776] T guttata [XP002189244]H sapiens [NP005525]andMmusculus [AAC52938]The alignment was generated with ClustalW andmodifiedmanually Amino acids conserved among all speciesare indicated as identical (lowast) highly conserved () or weakly conserved () The light yellow shade highlights the JAK2 binding site

liver and small intestine the transcriptional level of IFNGR1in 1-week-old goslings was obviously lower compared withthat in goose embryos Furthermore in the liver and spleenthe IFNGR2 transcriptional level was obviously increasedwhile it was apparently decreased in the small intestine andharderian gland

37 Effect of R848 on Transcriptional Levels of Goose IFN120574and IFNGRs As shown in Figure 9 R848 caused a highlysignificant upregulation of goose IFN120574 (119875 lt 005) comparedto the PBS control but no significant change in expression ofIFNGR1 (119875 = 025) and IFNGR2 (119875 = 007) was detectedThese results indicated that the R848 agonist could activate


MKPYSPGTYMTVSTCVNISTNSCDLSREVKETFSPYWFRVKAVVGSEQSEYVETNEFILQKHGKIGPPKLDLSRHADKII

VDIYHPVFPSMEFQPWITDNLDFMYKVTFWDNETQRKEEVFAEDCQVDKCSLDIPVTPNGSIYCVSAKSSLFENLIVGAP

SEESCIQVPLEQTTSTQNIVIVCVAVVIMGIILTLCCGFKKLRERNIKLPKSLVTVIRNLNMDNALESKSEGKYISIVSV

MPVQSALPLNSKEALLNIEPEEEAVSLDNFSEGASSCPPPEAPDKVEESSVQKITEEVPSDDEQNCKVKESYFISDSNQT

GISSNSSGPEVSATEIQQTVIPRSCPKFSGYDKPHVPLDMLIDVGEEQPVIAYRPTD

10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)

10MTSDWVESDPFVAERDTTIGPPKVNSVTVSSDSLLISVSPPFEFEEGTLQYHVSYWENATTTTKEMLVNNALFKIENLKQ

MTLYCFTIEIELKMHLYDRIPGLQRIPECYRTPMSETTRAAYIITTFTLVGLVLILIIIGLFCLWRHHKTIKYLCQPPLK100 130120 160140 150110

IPSHIEEYLRDPGMPHLEALENYHEEAPHDSLSVLCFEEGSEAYDDTLDGNTRSHSSSGDCEVT

170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90

120572-helix120573-sheet

(b)

Figure 5 Secondary structures of goose IFNGR1 and IFNGR2 Secondary structures of goose IFNGR1 and IFNGR2 were analyzed using theI-TASSER online server Both 120572-helices and 120573-sheets are shown in corresponding positions above the sequence

IFN120574 but did not affect the expression of IFNGR1 or IFNGR2in geeseThe results abovemay facilitate further studies of thegoose IFNGR-mediated immunological signaling pathway

4 Discussion

IFN120574 is a pleiotropic cytokine secreted by T-helper-1 (Th1)cells promoting both innate and adaptive responses toinfection within the host [14 15] The major producersof this cytokine are activated T cells natural killer (NK)cells and professional antigen-presenting cells (APCs) [16ndash18] IFN120574 binds to constitutively expressed IFN120574 recep-tors a heterodimer consisting of two chains IFNGR1 andIFNGR2 which then activates the downstream JAK-STATsignaling pathway The phosphorylated STAT proteins moveinto the nucleus bind specific DNA response elementsand directly transcribe IFN-stimulated genes to induce anantiviral immune response As reviewed elsewhere [19] IFN120574can contribute to the protection against infection with someviruses such as hepatitis B virus herpes simplex virus andlymphocytic choriomeningitis virus The antiviral responses

may rely on the expression levels of IFNGR1 and IFNGR2 aswell as the interaction between IFNGR and IFN120574

Until now comparatively little was known about avianIFN120574 receptors at the molecular level other than those ofchickens Herein we described the molecular cloning ofgoose IFNGR1 (1322 bp) and IFNGR2 (1438 bp) cDNA for thefirst time Goose IFNGR1 and IFNGR2 were found to bothpossess a TM region which demonstrated that they are singlemembrane proteins The JAK1 binding site (positions 209ndash214) and STAT1 binding site (positions 351ndash355) of IFNGR1were localized to the intracellular region which can recruitJAK1 and STAT1 for signal transduction The amino acids ofthese binding sites in birds have been reported to be relativelyconserved in both humans and mice [3] Similarly the JAK2binding site (positions 158ndash169) of IFNGR2 was also locatedat the intracellular region These specific motifs are relativelyconservative between birds and mammals [3]

In this study the goose IFNGR1 and IFNGR2 amino acidsequences were analyzed at the structural and phylogeneticlevels Prior to this study the secondary structures of IFNreceptors of geese were largely unknown We found that


Anas platyrhynchos XP005017811

Alligator sinensis XP006030325

Pelodiscus sinensis XP006112786

Chrysemys picta bellii XP005280397Ophiophagus hannah ETE71008Xenopus tropicalis AAI67921

Anolis carolinensis AGU16999Rattus norvegicus EDL93784

Mus musculus EDL03452

Papio anubis XP003898168

Macaca mulatta NP001253229

Homo sapiens AAH05333

Bos taurus AAI03297

Ovis aries XP004011420

Camelus ferus XP006188549

Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690

Taeniopygia guttata XP002189244

Geospiza fortis XP005427852

Zonotrichia albicollis XP005497041Crotalus horridus JAA96806

Anolis carolinensis AGL76447

Bos taurus AAS59772


Sus scrofa NP001104728

Mus musculus AAC52938

Rattus norvegicus NP001101783

Callicebus moloch ACA51056

Papio anubis ABW97193

Homo sapiens NP005525

Pan troglodytes JAA36651Danio rerio AAI63407

Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859

Falco peregrinus XP005230295

Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100

Figure 6 Phylogenetic analysis based on IFNGR1 and IFNGR2 amino acidsThe phylogenetic tree of partial vertebrate IFNGR1 and IFNGR2amino acid sequences was constructed using the NJ method in MEGA5 Numbers at branch nodes indicate the confidence level with 1000bootstrap replications IFNGR1 and IFNGR2 of birds are indicated with a green oval and those of geese are indicated with red circles

the secondary structure of the goose IFNGR1 protein con-tained 56 120572-helices 236 120573-sheets and 708 randomcoils which was different from that predicted for thecounterpart protein in chickens [7] Additionally the gooseIFNGR2 protein contained 98 120572-helices 286 120573-sheetsand 616 random coils suggesting certain differencesbetween the secondary structure of this protein in geeseand chickens [8] These results may aid in clarifying the

tertiary structures of goose IFNGR1 and IFNGR2 Differ-ences in secondary structures between IFNGR1 and IFNGR2may result in subtle changes of the higher order structuresand endow them with different functions Additionally theminimal divergence of IFNGR between geese and ducksfurther indicated the conservation of goose IFNGR1 andIFNGR2 during the evolution of waterfowl The structuraland evolutionary approaches to studying immune genes such


IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80

TissuesB BF CE CT GI H HG K LI LU PR SI SP T



Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574

Figure 7 Tissue distribution of IFN120574 IFNGR1 and IFNGR2 in goslings Tissues of three goslings (1 week of age) were collected and mRNAlevels of IFN120574 IFNGR1 and IFNGR2 (normalized to 120573-actin) were quantified by RT-qPCR Data are represented as the mean plusmn SEM (119899 = 3)Cecal tonsil CT liver Li lung Lu kidney K harderian gland HG brain B bursa of Fabricius BF cecum CE heart H small intestine SIspleen Sp thymus T gizzard Gi and proventriculus Pr

as IFNGR will also help us to unravel interspecies similaritiesand differences in host defense

Analysis of the tissue distribution of IFN120574 IFNGR1 andIFNGR2 in goslings showed that these genes were constitu-tively and widely expressed in different tissues Notably theresults showed that expression patterns of IFN120574 IFNGR1 andIFNGR2 were not completely the same in different periods ofdevelopment IFN120574 was widely detected in various samplesbut the level of IFN120574 in the brain of goslings was lowestSimilarly IFNGR1 and IFNGR2 were found at relatively lowlevels in the brain The main reason for these observationsmay be that the brain does not participate in the IFN-mediated immune response or lacks immune cells Chickensinfected with infectious bursal disease virus have shownextensive viral replication in the bursa and cecal tonsilswith an associated accumulation of T cells [20] In thisstudy both IFNGR1 and IFNGR2 were readily detected inthe cecal tonsil of goslings A possible explanation for thisphenomenon is that abundant lymphocytes accumulate in thececal tonsil which is responsible for the intestinal antiviralimmune responseThe abundant expression of goose IFNGR1and IFNGR2 in the cecal tonsil may contribute to the strongintestinal mucosal immunity Notably as shown in Figure 7

IFNGR1 and IFNGR2 levels in the lung were also relativelyhigher than those in the kidney and heart which may beattributed to alveolar macrophages as being the predominantcells in the lung As a result the lungs can secrete a large num-ber of bioactive cytokines which subsequently participate inthemucosal immune defense In addition IFN120574 and IFNGR2were observed to be widely expressed in the immune-relatedtissues including the bursa of Fabricius cecum spleen andthymus while IFNGR1 was extensively expressed in the bursaof Fabricius and cecal tonsil The similar tissue distributionof goose IFN120574 and its cognate receptors suggested that thesecytokines are immune-associated factors To some extent theinduction of the IFN120574 immune response may be reasonablyconnected with its associated receptors due to the similartissue-specific expression patterns

In order to explore the expression patterns during goosedevelopmental period we detected levels of IFN120574 IFNGR1and IFNGR2 in goose embryos and adult geese In the spleenthe expression of IFN120574 increased from embryos to goslingduring the early developmental period which is consistentwith prior observations of chicken IFN120574 [21] The decreaseof IFN120574 was observed in adult geese One of the possiblereasons for the reduction of IFN120574 may be the functional


0369

121530

35

40

45

50

Adult

TissuesBF CE H HG K LI LU SI SP T

Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W

Figure 8 Age-related mRNA expression analysis of goose IFN120574 and IFNGRs Comparative mRNA sequence analysis of goose IFN120574 and itsreceptors in certain tissues of embryos at EID20 goslings (1 week of age) and adult geese (3 months of age) 120573-actin was amplified as aninternal control Data are represented as the mean plusmn SEM (119899 = 3) Spleen Sp thymus T bursa of Fabricius BF harderian gland HG smallintestine SI heart H liver Li lung Lu kidney K and brain B

degeneration of the spleen in adult geese The expressionof IFNGR2 also showed a downward trend in the spleenwhile that of IFNGR1 did not which differed from expressionpatterns of these genes in the harderian gland These geneswere expressed in an organ-specific manner which wassimilar to the concept of tissue-specific innate immune geneexpression profiles [21ndash24] As both IFNGR1 and IFNGR2 arepotentially TM proteins their expression patterns remainedstable in most tissues Finally the defective productionof IFN120574 may be compensated by the high expression ofIFNGR2 in the adult period of development thus keepinga certain balance of the effectiveness of IFN120574 in the hostdefense system However in the cecum and small intestineIFN120574 IFNGR1 and IFNGR2 were detected at extremelyhigh levels during the embryonic stage but they declinedgradually during goose development Previous studies haddemonstrated that IFN120574 directly affected the barrier function

in model intestinal epithelial monolayers [25] Receptors forIFN120574 have been reported on the surface of epithelial cells [26]and endothelial cells [27] Thus observing high expressionlevels of goose IFN120574 and its associated receptors in thececum and small intestine in this study was reasonable Theresults also indicated that the IFN120574 immune system may beestablished during the embryonic stage Furthermore underunstimulated conditions no specific correlation between theexpression patterns of IFN120574 IFNGR1 and IFNGR2 in thesame tissue was observed Intriguingly low expression ofIFNGR1 in goslings may have been compensated by IFN120574and IFNGR2 These differences in the expression patternsof IFN120574 and its receptors in geese to some extent mayhave been simply caused by the functional compensation ofthesemolecules in different organs Furthermore R848 couldsignificantly upregulate IFN120574 but it did not influence theexpression of IFNGR1 and IFNGR2 by 10 h after stimulation


0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848

IFNGR1 IFNGR2 IFN120574

lowast

Figure 9 Effect of R848 on transcriptional levels of goose IFN120574 andIFNGRs The relative mRNA levels of IFNGR1 IFNGR2 and IFN120574at 10 h after stimulation of goose MNCs with R848 Each mRNAexpression value was normalized by 120573-actin Data are presented asthe mean plusmn SEM (119899 = 4) and differences between agonist-treatedcells and mock-treated cells were analyzed by the two-tailed 119905-testlowast119875 lt 005

Altogether these findings will expand our knowledge ofIFNGR-mediated immune responses in waterfowl

5 Conclusion

In summary we have identified and characterized IFN120574receptors in geese for the first time providing new insightsinto these immune molecules in this species Goose IFN120574and its receptors were found to be transcribed primarily inimmune-related tissues but the overall age-related expres-sion of goose IFN120574 IFNGR1 and IFNGR2 did not appear tobe directly correlated Furthermore R848 could significantlyinduce IFN120574 but not IFNGR1 or IFNGR2Neverthelessmuchwork is still needed to clarify the interaction between gooseIFN120574 and IFNGR1 or IFNGR2 which will contribute toa better understanding of the antiviral defense system ofaquatic birds

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Hao Zhou and Shun Chen contributed equally as co-firstauthors of this work

Acknowledgments

This work was funded by grants from the National Nat-ural Science Foundation of China (31201891) The PhDPrograms Foundation of Ministry of Education of China(20125103120012) the Sichuan Provincial Cultivation Pro-gram for Leaders of Disciplines in Science (2012JQ0040) theMajor Project of Education Department in Sichuan Province

(12ZA107) the Innovative Research Team Program in Edu-cation Department of Sichuan Province (nos 12TD0052013TD0015) the National Science and Technology SupportProgram (2015BAD12B05) the National Special Fund forAgro-Scientific Research in the Public Interest (201003012)and China Agricultural Research System (CARS-43-8)

References

[1] K Schroder P J Hertzog T Ravasi and D A Hume ldquoInter-feron-120574 an overview of signals mechanisms and functionsrdquoJournal of Leukocyte Biology vol 75 no 2 pp 163ndash189 2004

[2] B Saha S Jyothi Prasanna B Chandrasekar and D NandildquoGenemodulation and immunoregulatory roles of Interferon120574rdquoCytokine vol 50 no 1 pp 1ndash14 2010

[3] E A Bach M Aguet and R D Schreiber ldquoThe IFNgammareceptor a paradigm for cytokine receptor signalingrdquo AnnualReview of Immunology vol 15 pp 563ndash591 1997

[4] J E Darnell Jr I M Kerr and G R Stark ldquoJak-STAT pathwaysand transcriptional activation in response to IFNs and otherextracellular signaling proteinsrdquo Science vol 264 no 5164 pp1415ndash1421 1994

[5] M A Farrar and R D Schreiber ldquoThemolecular cell biology ofinterferon-120574 and its receptorrdquo Annual Review of Immunologyvol 11 pp 571ndash611 1993

[6] C A Bonjardim P C P Ferreira and E G Kroon ldquoInterferonssignaling antiviral and viral evasionrdquo Immunology Letters vol122 no 1 pp 1ndash11 2009

[7] X Han T Chen and M Wang ldquoMolecular cloning and char-acterization of chicken interferon-120574 receptor 120572-chainrdquo Journalof Interferon and Cytokine Research vol 28 no 7 pp 445ndash4532008

[8] C-L Han W Zhang H-T Dong X Han and M Wang ldquoAnovel gene of 120573 chain of the IFN-120574 receptor of Huiyang chickencloning distribution and CD assayrdquo Journal of Interferon andCytokine Research vol 26 no 7 pp 441ndash448 2006

[9] A J Karpala A Bagnaud-Baule K E Goossens J W Lowen-thal and A G D Bean ldquoOntogeny of the interferon system inchickensrdquo Journal of Reproductive Immunology vol 94 no 2pp 169ndash174 2012

[10] E Bar-Shira D Sklan and A Friedman ldquoEstablishment ofimmune competence in the avian GALT during the immediatepost-hatch periodrdquo Developmental and Comparative Immunol-ogy vol 27 no 2 pp 147ndash157 2003

[11] H Zhou S Chen M Wang and A Cheng ldquoInterferons andtheir receptors in birds a comparison of gene structure phylo-genetic analysis and cross modulationrdquo International Journal ofMolecular Sciences vol 15 no 11 pp 21045ndash21068 2014

[12] B Muhire D P Martin J K Brown et al ldquoA genome-widepairwise-identity-based proposal for the classification of virusesin the genus Mastrevirus (family Geminiviridae)rdquo Archives ofVirology vol 158 no 6 pp 1411ndash1424 2013

[13] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[14] U Boehm T Klamp M Groot and J C Howard ldquoCellularresponses to interferon-gammardquo Annual Review of Immunol-ogy vol 15 pp 749ndash795 1997


[15] S Pestka C D Krause and M R Walter ldquoInterferonsinterferon-like cytokines and their receptorsrdquo ImmunologicalReviews vol 202 no 1 pp 8ndash32 2004

[16] L A Lieberman and C A Hunter ldquoRegulatory pathwaysinvolved in the infection-induced production of IFN-120574 by NKcellsrdquoMicrobes and Infection vol 4 no 15 pp 1531ndash1538 2002

[17] J R Schoenborn and C B Wilson ldquoRegulation of interferon-120574 during innate and adaptive immune responsesrdquo Advances inImmunology vol 96 pp 41ndash101 2007

[18] D M Frucht T Fukao C Bogdan H Schindler J J OrsquoSheaand S Koyasu ldquoIFN-120574 production by antigen-presenting cellsmechanisms emergerdquo Trends in Immunology vol 22 no 10 pp556ndash560 2001

[19] R Shtrichman and C E Samuel ldquoThe role of gamma interferonin antimicrobial immunityrdquo Current Opinion in Microbiologyvol 4 no 3 pp 251ndash259 2001

[20] N Tanimura and J M Sharma ldquoAppearance of T cells in thebursa of Fabricius and cecal tonsils during the acute phaseof infectious bursal disease virus infection in chickensrdquo AvianDiseases vol 41 no 3 pp 638ndash645 1997

[21] M F Abdul-Careem D B Hunter M D Lambourne J Bartaand S Sharif ldquoOntogeny of cytokine gene expression in thechicken spleenrdquo Poultry Science vol 86 no 7 pp 1351ndash13552007

[22] A Lammers W H Wieland L Kruijt et al ldquoSuccessive immu-noglobulin and cytokine expression in the small intestine ofjuvenile chickenrdquo Developmental amp Comparative Immunologyvol 34 no 12 pp 1254ndash1262 2010

[23] S S ReemersD vanLeenenM JGrootKoerkamp et al ldquoEarlyhost responses to avian influenza A virus are prolonged andenhanced at transcriptional level depending on maturation ofthe immune systemrdquo Molecular Immunology vol 47 no 9 pp1675ndash1685 2010

[24] D Schokker A J W Hoekman M A Smits and J M J RebelldquoGene expression patterns associated with chicken jejunaldevelopmentrdquo Developmental and Comparative Immunologyvol 33 no 11 pp 1156ndash1164 2009

[25] J L Madara and J Stafford ldquoInterferon-120574 directly affectsbarrier function of cultured intestinal epithelial monolayersrdquoThe Journal of Clinical Investigation vol 83 no 2 pp 724ndash7271989

[26] U Ucer H Bartsch P Scheurich and K Pfizenmaier ldquoBio-logical effects of 120574-interferon on human tumor cells quantityand affinity of cell membrane receptors for 120574-IFN in relationto growth inhibition and induction of HLA-DR expressionrdquoInternational Journal of Cancer vol 36 no 1 pp 103ndash108 1985

[27] A H Stoplen E C Guinan W Fiers and J S Pober ldquoRecom-binant tumor necrosis factor and immune interferon act singlyand in combination to reorganize human vascular endothelialcell monolayersrdquo The American Journal of Pathology vol 123no 1 pp 16ndash24 1986


recognition receptors (PRRs) has indicated that the IFN sys-tem is somewhat immature during the early developmentalstage of chick embryonic cells [9] The development of IFN120574in the intestinal immunity of juvenile chickens has beencharacterized as well [10]

Based on a comprehensive review of reports on the genestructure evolutionary analysis and crosstalk between IFNand its cognate receptors in birds [11] studies of the IFNsystem in waterfowl appear to be lagging behind In additionthe development and immune characteristics of avian IFN120574are still poorly understood Moreover the duck IFNGR1 andIFNGR2 genes are only predicted sequences Up to now noinformation has been made available on the identification ofgoose IFN receptors Given these considerations this studywas conducted to examine the expression level of goose IFN120574and its associated receptors throughout the embryogenesisphase and posthatch period Herein for the first time gooseIFNGR1 and IFNGR2 cDNA sequences were identified andthe corresponding amino acid sequences as well as structuralcharacteristics were analyzed Comparative analysis of gooseIFNGR sequences with those in birds mammals fish andreptiles may shed light on the evolutionary position of goosegenes among vertebrates The tissue distribution and age-related expression of goose IFN120574 and IFN120574 receptors alsowere analyzed in this study The results of this study willextend existing information on the age-related developmentof goose IFN120574 and its cognate receptors which may shedfurther light on IFN antiviral responses in this species

2 Methods

21 Animals The study was conducted with Sichuan WhiteGeese (Chinese goose A cygnoides) Goose embryos at 20embryonic incubation days (EID20) goslings (1 week of age)and adult geese (3 months of age) were chosen All animalsin this study were purchased from the farm at SichuanAgricultural University (Yarsquoan city Sichuan province) One-week-old goslings and adult geese were maintained for 3days in laboratory animal rooms for acclimation prior toexperiments and water and fodder were provided Thewelfare of the animals was ensured during the samplingprocess

22 RNA Extraction and cDNA Synthesis The birds wereeuthanized and then tissues were collected and snap-frozenin liquid N

2 The chosen tissues included cecal tonsil liver

lung kidney harderian gland brain bursa of Fabriciuscecum heart small intestine spleen thymus gizzard andproventriculus Total RNAwas extracted from various tissuesusing Trizol reagent (Invitrogen Carlsbad CA USA) accord-ing to themanufacturerrsquos instructionsThe cDNAwas synthe-sized using the QuantScript RT kit (Promega Madison WIUSA) according to the manufacturerrsquos instructions FinallycDNA templates of all different samples were stored at minus80∘Cuntil use

23 Molecular Cloning of Goose IFNGR A partial sequenceof goose IFNGR was amplified by the degenerate primersF1 R1 F2 and R2 (all primer sequences used in this

study are listed in Table 1) which were designed based onthe conserved regions among its counterparts in birds (allreference sequences used in this study are listed in Table 2)The resultant PCR fragments were subcloned into the pGEM-T Easy Vector (Promega) followed by transformation ofDH5120572 cells The positive clones were sequenced by using theABI 3730 XL sequencer (Applied Biosystems Foster CityCA USA) Subsequently 31015840 and 51015840 rapid amplification ofcDNA ends (RACE) was performed to obtain the full-lengthcDNA sequence of target genes Based on the partial sequenceobtained Gene Specific Primers (GSPs) including 3GSP13GSP2 5GSP1 5GSP2 and 5GSP3 were designed to obtainthe full-length goose IFNGR cDNA For 31015840-RACE the firststrand cDNAwas synthesized using theAdapter Primer (AP)The 31015840-end of goose IFNGR was amplified by nested PCRusing the primers 3GSP1 and 3GSP2 with AP1 and AP2 For51015840-RACE the first strand cDNAwas synthesized by using theprimer 5GSP1 andM-MLVReverse Transcriptase (Promega)A homopolymeric tail was then added to the 31015840-end of thecDNA using TdT and dCTP (TaKaRa Kyoto Japan) The51015840-end of goose IFNGR was also obtained by two nestedPCRs with the primer pairs 5GSP2Abridged Anchor Primer(AAP) and 5GSP3Abridged Universal Amplification Primer(AUAP) Finally the full-length coding sequence of gooseIFNGR was amplified by using Primer STAR Max DNApolymerase (TaKaRa)

24 BioinformaticAnalysis of Sequences Potential open readingframes (ORFs) were analyzed by using the ORF finderprogram (httpwwwncbinlmnihgovgorfgorfhtml) andtranslated into the corresponding amino acids using DNA-MAN N-Glycosylation sites were predicted with onlinesoftware (httpwwwcbsdtudkservicesNetNGlyc) After-wards the TM region was examined with the TMHMMserver version 20 (httpwwwcbsdtudkservicesTMHMM)The potential protein domains of amino acid sequences wereforecasted via the SMART server (httpsmartembl-heidel-bergde) Pairwise identity analysis was performed withthe Species Demarcation Tool [12] Alignment of putativeamino acid sequences of IFNGR1 and IFNGR2 was per-formed using the Clustal program and sequence similaritieswere calculated with the MegAlign program Secondarystructures were analyzed using the I-TASSER program(httpzhanglabccmbmedumichedu) To analyze the evo-lutionary relationships between type II IFN receptors in birdsand other vertebrates a phylogenetic tree was constructedusing amino acid sequences via the neighbor-joining (NJ)method in MEGA4 with bootstrap analysis based on 1000repetitions [13]

25 Tissue Distribution and Age-Related Expression Analysisof Goose IFNGR mRNA The tissue distribution of IFNGRin healthy 1-week-old goslings was studied by real-timequantitative qPCR (RT-qPCR) using the Bio-Rad CFX96Real-Time Detection System The age-related expressionanalysis of goose IFN120574 receptors at the mRNA level incertain tissues of geese (embryonic incubation 20 days andadult) was also detected by RT-qPCR Where possible theprimers were designed across intron and extron boundaries












5RACE-GSP



Real-time PCR







IFNGR1



IFNGR2




2O




3 Results































1911181222715236182451112541142631172721202811232901262991292108132211713521261

























1911812711361284515854188631118721148811

901178

991208

10811171

13511261

(b)

1 377199177

TM

(c)

1

TM

22414597 12319

FN3

(d)



100279 100315 875 10031 632 595 100

307 598 587 588 100333 584 594 576 897 100318 602 557 54 806 842 100325 58 574 536 784 814 799 100292 591 572 552 799 78 763 776 100308 627 602 586 609 628 593 611 61314 666 635 61 713 696 685 678 706312 616 563 575 596 597 566 588 607313 609 558 563 594 593 565 583 608343 457 449 458 483 461 467 442 484323 455 454 458 45 451 453 444 42529 404 39 418 429 436 433 431 436

269 304 319 281 289 311 318 293 31230 296 319 295 306 318 329 314 286

316 321 338 352 327 335 346 313 351321 323 338 357 327 333 344 313 351326 323 322 344 325 335 348 329 354313 349 359 353 344 358 378 364 35274 341 33 325 304 315 332 34 32306 322 323 335 319 321 337 298 325309 333 337 337 309 352 34 316 31528 314 325 348 363 344 368 306 34

321 352 325 285 289 295 302 289 274195 257 253 257 224 224 229 222 226235 219 24 228 228 254 235 231 215

100683 100631 729 100627 715 964 100456 487 457 45 100441 474 455 447 723 100427 467 441 447 508 504 100301 292 305 307 319 301 311 100296 301 298 294 36 317 343 74 100317 33 336 345 374 353 378 502 533 100315 33 336 345 374 35 376 502 538 994 100308 323 333 343 371 348 38 511 535 953 951 100348 365 359 356 408 383 384 492 474 616 621 623 100299 329 325 335 36 355 345 495 492 607 611 603 755 100319 319 311 315 365 331 373 496 487 609 613 612 726 73 100316 34 333 345 359 352 363 509 483 591 596 597 715 702 951 100332 339 322 32 377 346 328 291 308 325 323 337 317 308 284 288323 311 30 301 327 313 286 282 283 319 31 311 299 285 282 276229 248 245 234 251 264 217 211 226 21 21 202 231 232 215 225248 207 233 241 233 239 256 225 241 245 241 239 222 238 236 23

100401 100193 204 100237 205 258 100


(a)

100471 100302 374 100308 347 797 100346 34 748 747 100282 272 578 602 525 100326 368 488 498 509 408 100342 40 477 512 578 41 711 100363 387 51 521 521 413 721 748 100369 39 51 528 521 417 723 752 91329 353 464 441 498 448 578 603 622

315 364 435 459 459 356 59 632 627352 382 50 483 511 421 629 657 656307 343 317 324 308 246 328 377 355306 353 366 349 34 284 344 362 36533 361 335 308 329 254 335 37 343

317 36 335 312 312 254 332 366 34326 365 339 315 319 25 338 374 34234 367 347 309 321 262 318 379 362

303 31 303 302 286 235 333 351 357306 355 327 315 311 256 339 356 36931 35 291 316 298 261 29 34 324

268 285 268 277 286 262 279 288 285

100622 100614 844 100661 677 61 100361 338 345 337 100369 368 356 338 815 100344 301 34 352 605 599 100352 311 341 353 606 594 958 100342 311 349 354 614 605 952 988 100352 301 327 35 604 591 862 863 865 100353 294 337 333 518 504 637 644 651 626 100366 295 34 314 523 519 636 645 652 635 931 100335 317 333 332 548 548 651 651 655 64 772 755 100297 255 295 314 272 278 27 276 284 286 254 262 285 100


(b)









--------------------------------------------------


------------------------------------MGAPLALMVLTALV


--------------------------------------MALLFLLPLVMQ



--------------------------------------------------
























































(a)

Figure 3 Continued
















(b)



































10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)




170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90


(b)



4 Discussion















Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References








































5RACE-GSP



Real-time PCR







IFNGR1



IFNGR2




2O




3 Results































1911181222715236182451112541142631172721202811232901262991292108132211713521261

























1911812711361284515854188631118721148811

901178

991208

10811171

13511261

(b)

1 377199177

TM

(c)

1

TM

22414597 12319

FN3

(d)



100279 100315 875 10031 632 595 100

307 598 587 588 100333 584 594 576 897 100318 602 557 54 806 842 100325 58 574 536 784 814 799 100292 591 572 552 799 78 763 776 100308 627 602 586 609 628 593 611 61314 666 635 61 713 696 685 678 706312 616 563 575 596 597 566 588 607313 609 558 563 594 593 565 583 608343 457 449 458 483 461 467 442 484323 455 454 458 45 451 453 444 42529 404 39 418 429 436 433 431 436

269 304 319 281 289 311 318 293 31230 296 319 295 306 318 329 314 286

316 321 338 352 327 335 346 313 351321 323 338 357 327 333 344 313 351326 323 322 344 325 335 348 329 354313 349 359 353 344 358 378 364 35274 341 33 325 304 315 332 34 32306 322 323 335 319 321 337 298 325309 333 337 337 309 352 34 316 31528 314 325 348 363 344 368 306 34

321 352 325 285 289 295 302 289 274195 257 253 257 224 224 229 222 226235 219 24 228 228 254 235 231 215

100683 100631 729 100627 715 964 100456 487 457 45 100441 474 455 447 723 100427 467 441 447 508 504 100301 292 305 307 319 301 311 100296 301 298 294 36 317 343 74 100317 33 336 345 374 353 378 502 533 100315 33 336 345 374 35 376 502 538 994 100308 323 333 343 371 348 38 511 535 953 951 100348 365 359 356 408 383 384 492 474 616 621 623 100299 329 325 335 36 355 345 495 492 607 611 603 755 100319 319 311 315 365 331 373 496 487 609 613 612 726 73 100316 34 333 345 359 352 363 509 483 591 596 597 715 702 951 100332 339 322 32 377 346 328 291 308 325 323 337 317 308 284 288323 311 30 301 327 313 286 282 283 319 31 311 299 285 282 276229 248 245 234 251 264 217 211 226 21 21 202 231 232 215 225248 207 233 241 233 239 256 225 241 245 241 239 222 238 236 23

100401 100193 204 100237 205 258 100


(a)

100471 100302 374 100308 347 797 100346 34 748 747 100282 272 578 602 525 100326 368 488 498 509 408 100342 40 477 512 578 41 711 100363 387 51 521 521 413 721 748 100369 39 51 528 521 417 723 752 91329 353 464 441 498 448 578 603 622

315 364 435 459 459 356 59 632 627352 382 50 483 511 421 629 657 656307 343 317 324 308 246 328 377 355306 353 366 349 34 284 344 362 36533 361 335 308 329 254 335 37 343

317 36 335 312 312 254 332 366 34326 365 339 315 319 25 338 374 34234 367 347 309 321 262 318 379 362

303 31 303 302 286 235 333 351 357306 355 327 315 311 256 339 356 36931 35 291 316 298 261 29 34 324

268 285 268 277 286 262 279 288 285

100622 100614 844 100661 677 61 100361 338 345 337 100369 368 356 338 815 100344 301 34 352 605 599 100352 311 341 353 606 594 958 100342 311 349 354 614 605 952 988 100352 301 327 35 604 591 862 863 865 100353 294 337 333 518 504 637 644 651 626 100366 295 34 314 523 519 636 645 652 635 931 100335 317 333 332 548 548 651 651 655 64 772 755 100297 255 295 314 272 278 27 276 284 286 254 262 285 100


(b)









--------------------------------------------------


------------------------------------MGAPLALMVLTALV


--------------------------------------MALLFLLPLVMQ



--------------------------------------------------
























































(a)

Figure 3 Continued
















(b)



































10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)




170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90


(b)



4 Discussion















Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References


























































1911181222715236182451112541142631172721202811232901262991292108132211713521261

























1911812711361284515854188631118721148811

901178

991208

10811171

13511261

(b)

1 377199177

TM

(c)

1

TM

22414597 12319

FN3

(d)



100279 100315 875 10031 632 595 100

307 598 587 588 100333 584 594 576 897 100318 602 557 54 806 842 100325 58 574 536 784 814 799 100292 591 572 552 799 78 763 776 100308 627 602 586 609 628 593 611 61314 666 635 61 713 696 685 678 706312 616 563 575 596 597 566 588 607313 609 558 563 594 593 565 583 608343 457 449 458 483 461 467 442 484323 455 454 458 45 451 453 444 42529 404 39 418 429 436 433 431 436

269 304 319 281 289 311 318 293 31230 296 319 295 306 318 329 314 286

316 321 338 352 327 335 346 313 351321 323 338 357 327 333 344 313 351326 323 322 344 325 335 348 329 354313 349 359 353 344 358 378 364 35274 341 33 325 304 315 332 34 32306 322 323 335 319 321 337 298 325309 333 337 337 309 352 34 316 31528 314 325 348 363 344 368 306 34

321 352 325 285 289 295 302 289 274195 257 253 257 224 224 229 222 226235 219 24 228 228 254 235 231 215

100683 100631 729 100627 715 964 100456 487 457 45 100441 474 455 447 723 100427 467 441 447 508 504 100301 292 305 307 319 301 311 100296 301 298 294 36 317 343 74 100317 33 336 345 374 353 378 502 533 100315 33 336 345 374 35 376 502 538 994 100308 323 333 343 371 348 38 511 535 953 951 100348 365 359 356 408 383 384 492 474 616 621 623 100299 329 325 335 36 355 345 495 492 607 611 603 755 100319 319 311 315 365 331 373 496 487 609 613 612 726 73 100316 34 333 345 359 352 363 509 483 591 596 597 715 702 951 100332 339 322 32 377 346 328 291 308 325 323 337 317 308 284 288323 311 30 301 327 313 286 282 283 319 31 311 299 285 282 276229 248 245 234 251 264 217 211 226 21 21 202 231 232 215 225248 207 233 241 233 239 256 225 241 245 241 239 222 238 236 23

100401 100193 204 100237 205 258 100


(a)

100471 100302 374 100308 347 797 100346 34 748 747 100282 272 578 602 525 100326 368 488 498 509 408 100342 40 477 512 578 41 711 100363 387 51 521 521 413 721 748 100369 39 51 528 521 417 723 752 91329 353 464 441 498 448 578 603 622

315 364 435 459 459 356 59 632 627352 382 50 483 511 421 629 657 656307 343 317 324 308 246 328 377 355306 353 366 349 34 284 344 362 36533 361 335 308 329 254 335 37 343

317 36 335 312 312 254 332 366 34326 365 339 315 319 25 338 374 34234 367 347 309 321 262 318 379 362

303 31 303 302 286 235 333 351 357306 355 327 315 311 256 339 356 36931 35 291 316 298 261 29 34 324

268 285 268 277 286 262 279 288 285

100622 100614 844 100661 677 61 100361 338 345 337 100369 368 356 338 815 100344 301 34 352 605 599 100352 311 341 353 606 594 958 100342 311 349 354 614 605 952 988 100352 301 327 35 604 591 862 863 865 100353 294 337 333 518 504 637 644 651 626 100366 295 34 314 523 519 636 645 652 635 931 100335 317 333 332 548 548 651 651 655 64 772 755 100297 255 295 314 272 278 27 276 284 286 254 262 285 100


(b)









--------------------------------------------------


------------------------------------MGAPLALMVLTALV


--------------------------------------MALLFLLPLVMQ



--------------------------------------------------
























































(a)

Figure 3 Continued
















(b)



































10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)




170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90


(b)



4 Discussion















Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References






























100279 100315 875 10031 632 595 100

307 598 587 588 100333 584 594 576 897 100318 602 557 54 806 842 100325 58 574 536 784 814 799 100292 591 572 552 799 78 763 776 100308 627 602 586 609 628 593 611 61314 666 635 61 713 696 685 678 706312 616 563 575 596 597 566 588 607313 609 558 563 594 593 565 583 608343 457 449 458 483 461 467 442 484323 455 454 458 45 451 453 444 42529 404 39 418 429 436 433 431 436

269 304 319 281 289 311 318 293 31230 296 319 295 306 318 329 314 286

316 321 338 352 327 335 346 313 351321 323 338 357 327 333 344 313 351326 323 322 344 325 335 348 329 354313 349 359 353 344 358 378 364 35274 341 33 325 304 315 332 34 32306 322 323 335 319 321 337 298 325309 333 337 337 309 352 34 316 31528 314 325 348 363 344 368 306 34

321 352 325 285 289 295 302 289 274195 257 253 257 224 224 229 222 226235 219 24 228 228 254 235 231 215

100683 100631 729 100627 715 964 100456 487 457 45 100441 474 455 447 723 100427 467 441 447 508 504 100301 292 305 307 319 301 311 100296 301 298 294 36 317 343 74 100317 33 336 345 374 353 378 502 533 100315 33 336 345 374 35 376 502 538 994 100308 323 333 343 371 348 38 511 535 953 951 100348 365 359 356 408 383 384 492 474 616 621 623 100299 329 325 335 36 355 345 495 492 607 611 603 755 100319 319 311 315 365 331 373 496 487 609 613 612 726 73 100316 34 333 345 359 352 363 509 483 591 596 597 715 702 951 100332 339 322 32 377 346 328 291 308 325 323 337 317 308 284 288323 311 30 301 327 313 286 282 283 319 31 311 299 285 282 276229 248 245 234 251 264 217 211 226 21 21 202 231 232 215 225248 207 233 241 233 239 256 225 241 245 241 239 222 238 236 23

100401 100193 204 100237 205 258 100


(a)

100471 100302 374 100308 347 797 100346 34 748 747 100282 272 578 602 525 100326 368 488 498 509 408 100342 40 477 512 578 41 711 100363 387 51 521 521 413 721 748 100369 39 51 528 521 417 723 752 91329 353 464 441 498 448 578 603 622

315 364 435 459 459 356 59 632 627352 382 50 483 511 421 629 657 656307 343 317 324 308 246 328 377 355306 353 366 349 34 284 344 362 36533 361 335 308 329 254 335 37 343

317 36 335 312 312 254 332 366 34326 365 339 315 319 25 338 374 34234 367 347 309 321 262 318 379 362

303 31 303 302 286 235 333 351 357306 355 327 315 311 256 339 356 36931 35 291 316 298 261 29 34 324

268 285 268 277 286 262 279 288 285

100622 100614 844 100661 677 61 100361 338 345 337 100369 368 356 338 815 100344 301 34 352 605 599 100352 311 341 353 606 594 958 100342 311 349 354 614 605 952 988 100352 301 327 35 604 591 862 863 865 100353 294 337 333 518 504 637 644 651 626 100366 295 34 314 523 519 636 645 652 635 931 100335 317 333 332 548 548 651 651 655 64 772 755 100297 255 295 314 272 278 27 276 284 286 254 262 285 100


(b)









--------------------------------------------------


------------------------------------MGAPLALMVLTALV


--------------------------------------MALLFLLPLVMQ



--------------------------------------------------
























































(a)

Figure 3 Continued
















(b)



































10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)




170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90


(b)



4 Discussion















Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References






























--------------------------------------------------


------------------------------------MGAPLALMVLTALV


--------------------------------------MALLFLLPLVMQ



--------------------------------------------------
























































(a)

Figure 3 Continued
















(b)



































10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)




170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90


(b)



4 Discussion















Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References












































(b)



































10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)




170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90


(b)



4 Discussion















Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References























































10 20 30 40 50 60 70 80

90 100 130120 160140 150110

170 180 210200 240220 230190

250 260 290280 320300 310270

330 340 370360350Goose IFNGR1

(a)




170 180 210200 220190Goose IFNGR2

20 30 40 50 60 70 80

90


(b)



4 Discussion















Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References







































Bos taurus AAI03297



Sus scrofa BAN

09008

Onc

orhy

nchu

s myk

iss A

BY87

189

Gal

lus g

allu

s AAV

6777

6

Melo

psitt

acus

und

ulat

us X

P005

1518

48

Falco

cher

rug X

P005

4386

64

Falco

per

egrin

us X

P005

2344

45

Goose

IFNGR2

Anas pla

tyrhy

nchos

XP0050

1390

3

Columba livia

XP005511438

Pseudopodoces

humilis XP005526690





Bos taurus AAS59772









Oncorhynchus m

ykiss NP001117888

Geo

spiz

a for

tis X

P005

4201

90

Zono

tric

hia a

lbic

ollis

XP0

0548

9284

Taen

iopy

gia g

utta

ta X

P002

1947

63Fi

cedu

la alb

icolli

s XP0

0504

3949

Pseu

dopo

doce

s hum

ilis X

P005

5187

07

Colum

ba liv

ia XP0

0550

9491

Gallus

gallu

s NP00

1123

859

Melopsitt

acus u

ndulatus X

P005154885

Falco ch

errug XP005435859


Goose IFNGR1

02

100

100

100

99

98

57

79

100

7978

87

66

98

9897

100

96

90

84

72

76

58

5784

68

99

24

83

29 4977

94

99

47

72 100

100

43

1610

0546396

98

71

99

9973

100





IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References






























IFNGR1

0

1

2

360

80

100

120

Relat

ive e

xpre

ssio

n

IFNGR2

0

2

4

620

40

60

80




Relat

ive e

xpre

ssio

n

0

2

4

6

8

10

12

14

Rela

tive e

xpre

ssio

n

IFN120574







0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References






























0369

121530

35

40

45

50

Adult


Rela

tive e

xpre

ssio

n

IFN120574

EID201W

IFNGR1

02468

10121416182022

Relat

ive e

xpre

ssio

n


AdultEID201W

IFNGR2

02468

101214161820

Relat

ive e

xpre

ssio

n


AdultEID201W





0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References






























0

02

04

06

08

1

12

Relat

ive e

xpre

ssio

n

PBSR848


lowast



5 Conclusion






Acknowledgments



References





























Identification of Type II Interferon Receptors in Geese: Gene ...

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