Characterisation of chicken viperin

Molecular Immunology 63 (2015) 373–380

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Characterisation of chicken viperin Kate E. Goossens a,b,1 , Adam J. Karpala a,∗,1 , Andreas Rohringer a , Alistair Ward b , Andrew G.D. Bean a a b

CSIRO Australian Animal Health Laboratory, Private Bag 24, Geelong, Victoria 3220, Australia School of Medicine, Deakin University, Waurn Ponds, Victoria, Australia

a r t i c l e

i n f o

Article history: Received 11 June 2014 Received in revised form 17 September 2014 Accepted 17 September 2014 Available online 11 October 2014 Keywords: Viperin Interferon Avian influenza Chicken

a b s t r a c t The identification of immune pathways that protect against pathogens may lead to novel molecular therapies for both livestock and human health. Interferon (IFN) is a major response pathway that stimulates multiple genes targeted towards reducing virus. Viperin is one such interferon stimulated gene (ISG) that helps protect mammals from virus and may be critical to protecting chickens in the same way. In chickens, ISGs are not generally well characterised and viperin, in concert with other ISGs, may be important in protecting against virus. Here we identify chicken viperin (ch-viperin) and show that ch-viperin is upregulated in response to viral signature molecules. We further show that viperin is upregulated in response to virus infection in vivo. This data will benefit investigators targeting the antiviral pathways in the chicken. Crown Copyright © 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction IFNs are rapidly induced following infection and subsequently activate hundreds of ISGs (de Veer et al., 2001; Sen and Sarkar, 2007). ISGs generally display a range of antiviral activities against diverse pathogens (Sadler and Williams, 2008; Sen, 2001). In mammals, the majority of ISGs remain largely uncharacterised with regard to their antiviral functions (Liu et al., 2011; Schoggins and Rice, 2011). However, our understanding of chicken ISG’s is further limited to a handful of characterised genes. It is important to investigate additional chicken ISG orthologues to broaden our understanding of innate immune pathways to promote protective strategies against virus in the chicken. Viperin, is an ISG that is highly conserved across a diversity of species, suggesting it may play an important role in the innate immune response of the chicken (Chin and Cresswell, 2001; Jiang et al., 2008). During the early stages of infection, mammalian viperin is rapidly expressed in response to a range of signals; including virus, IFN and microbial by products such as LPS and dsRNA (Brodsky et al., 2007). Viperin exhibits broad antiviral and antimicrobial activity, by mechanisms which are still being understood (Hinson et al., 2010). Thus far, viperin has been shown to inhibit viral protein and/or RNA

∗ Corresponding author at: Australian Animal Health Laboratory, CSIRO 5, Portarlington Rd, Geelong 3220, Australia. Tel.: +61 03 52275791; fax: +61 03 52275000. E-mail addresses: [email protected], [email protected] (A.J. Karpala). 1 These authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.molimm.2014.09.011 0161-5890/Crown Copyright © 2014 Published by Elsevier Ltd. All rights reserved.

biosynthesis for a number of different viruses (Chin and Cresswell, 2001; Jiang et al., 2010). In addition viperin has been shown to localise to the ER, which disrupts lipid rafts thereby inhibiting the trafficking of soluble virally encoded proteins during influenza infection (Hinson and Cresswell, 2009; Wang et al., 2007). To generate more effective treatment options for the control of poultry diseases, a deeper understanding of the chicken innate immune response will be helpful and viperin represents an interesting ISG to investigate in chickens, because of its involvement in response to a number of viral and bacterial infections in mammals. Therefore, the aim of this research was to identify and characterise the chicken viperin gene.

2. Materials and methods 2.1. Cell culture and bioassays Cells were cultured in a 37 ◦ C humidified incubator supplemented with 5% CO2 . Splenocytes were prepared as previously described (Karpala et al., 2008). Briefly, spleens were harvested from 3 to 4 week old specific-pathogen-free (SPF) chickens, and single cell suspensions obtained by dispersal through a 70 ␮m sieve (BD Falcon). The leukocyte suspensions were layered over 20 mL of lymphoprep (Nicomed Pharma) and prepared according to manufacturers’ instructions and maintained in DMEM supplemented with 10% FCS. Cells were treated with 10 ␮g/mL or transfected with 1 ␮g/mL of pIC (Invitrogen), to mimic dsRNA, or 10 ␮g/mL of LPS (Sigma–Aldrich) or 2 ␮g/mL of endotoxin free DNA (EF-DNA)

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Fig. 1. Identification of chicken viperin transcripts. (A) PCR cloning strategy for chicken viperin with the position of primers indicated (see Table 1 for primer sequences). (B) cDNA from chicken splenocytes, stimulated with chicken IFN-␣, was PCR amplified with viperin specific primers vip1R and vip1F and yielded a single product of ∼800 bp. (C, D) cDNA from chicken splenocytes stimulated with poly(I:C) was subjected to 5 RACE using vipOUT and the 5 RACE outer primer (C), followed by nested PCR with vipIN1 or vipIN2 primers paired with the 5 RACE outer primer (D) (M = DNA marker).

1

atgctgctgg gcgttctgga tcacctgccg ctggccctgg cgcgggcggt gctggcagcg M L L G V L D H L P L A L A R A V L A A

61

ctgcgcgggc ggctgagcgc gctgtgctgg gggctgacgc ccctcgttct gcctctgctc L R G R L S A L C W G L T P L V L P L L

121

tcctggaggc ggcggtcggg ccccgacacc cccgcggcac cccgggagga caaggacgag S W R R R S G P D T P A A P R E D K D E

181

acagttccga ctcccaccag cgtcaattat cacttcacca ggcagtgcaa ctacaagtgt T V P T P T S V N Y H F T R Q C N Y K C

241

ggtttctgct tccacacggc caagacctcc ttcgtgctgc ccctggagga agccaagcgg G F C F H T A K T S F V L P L E E A K R

301

gggctggcaa tgcttaagga ggcgggaatg gagaaaataa atttctcagg aggagaacca G L A M L K E A G M E K I N F S G G E P

361

tttcttcagg acagaggcga atttgtaggc cagctggtcc agttctgcaa agaggagctg D R G E F V G Q L V Q F C K E E L F L Q

421

aaactgccaa gcgtcagcat tgtgagcaat ggcagcctga tcagggaacg gtggttcaag S V S I V S N G S L I R E R W F K K L P

481

aagtatggtg aatatttaga cattctggca atttcatgtg atagttttaa tgaggaagtc E Y L D I L A I S C D S F N E E V K Y G

541

aacgttttaa ttggtcgtgg tcaaggaagg aagaaccatg tggaaaatct gcataaactg I G R G Q G R K N H V E N L H K L N V L

601

aggcagtggt gccgagatta tgctgttgct ttcaaaataa actccgtgat caacagattt C R D Y A V A F K I N S V I N R F R Q W

661

aatgttgagg aagatatgaa tgagcagatc aaggcactca atcctgtgcg ctggaaggtg E D M N E Q I K A L N P V R W K V N V E

721

ttccagtgcc taatcattga aggagagaac agtggggaag atgctctgag agaagcagat F Q C L I I E G E N S G E D A L R E A D

781

aaatttgtta tcagcgatga agactttgag caattcctgg aacgccacaa agatatctcc I S D E D F E Q F L E R H K D I S K F V

841

tgtttggtac ctgaatctaa tcagaagatg agagattcat acctcatttt ggatgaatat C L V P E S N Q K M R D S Y L I L D E Y

901

atgcgttttc taaactgtag aaacggacgg aaagagcctt ccaagtctat cctggatgtt L N C R N G R K E P S K S I L D V M R F

961

ggtgtagaag cggcaataaa attcagtgga tttgatgaga agatgttcct aaaacgagga A A I K F S G F D E K M F L K R G G V E

1021

ggaaagtatg tgtggagtaa agcagacatg attctggact ggtaa G K Y V W S K A D M I L D W -

Fig. 2. Nucleotide and predicted amino acid sequence of the chicken viperin gene. The final sequence was assembled from the PCR and 5 RACE PCR products presented in Fig. 1 and deposited into GenBank (Accession Number. EU427332), and is presented here with the predicted amino acid translation (arrows indicate intron locations).

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Table 1 Comparison of chicken viperin protein with those of other animals. Animals

Accession number

Ch.

Size (aa)

Identical amino acids (%)

Total amino acid similarity (%)

Turkey

ENSMGAT 00000015732 ENSTGUT 00000013531 ENSPSIT 00000007359 Q2HJF9 NP 542388 XP 515283 XP 851276 Q9MZU4 O70600 NP 067359 ENSACAT 00000007061 ABJ97316

2

354

96

99

3

291

92

98

?

347

76

90

11 2 ? 17 3 6 12 1

363 361 361 361 362 360 362 356

76 75 75 75 75 74 74 74

88 88 88 88 88 92 88 87

17

346

71

88

Zebra finch Chinese soft-shell turtle Cow Human Chimpanzee Dog Pig Rat Mouse Anole lizard Zebrafish

(Invitrogen) to mimic bacterial infection, or 1 mM of loxoribine (Invitrogen). Chicken IFN-␣ (GenWay) was used for IFN treatments. Cell transfections were carried out using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions.

infection. IBDV-infected birds were euthanised at either 2 or 4 days PI, then tissues collected and stored in RNA later for quantitative real time PCR (qRT-PCR) analysis. 2.3. RNA isolation and reverse transcription

2.2. Virus infections Influenza virus infections in chickens were previously carried out and described in Burggraaf et al. (2014). Briefly, obtained viruses were passed twice in chicken eggs then diluted 1:100 and inoculated through an oral–nasal–ocular route with a volume of 0.2 mL then tissues harvested after 24 h. For the IBDV infections three-week-old SPF chickens were infected with classical IBDV (01/09 = Australian classical IBDV strain) or variant IBDV (21/06 = Australian variant IBDV strain) by intraocular inoculation with 0.1 mL PBSA containing105 EID50 of virus. Chickens were housed in isolators and monitored for clinical signs of

RNA was harvested from tissue samples using Tri-reagent (Sigma–Aldrich) according to manufacturer’s instructions. RNA (2 ␮g) was DNase (Promega) treated according to manufacturer’s instructions then reverse transcribed (Promega) to produce cDNA as previously described (Karpala et al., 2008). 2.4. Semi-quantitative RT-PCR (qRT-PCR) The relative quantification of gene expression was analysed by qRT-PCR carried out on an ABI Prism 7700 sequence detection system and used the comparative cycle threshold (CT) method,

Fig. 3. Genomic organization of chicken viperin locus. (A) Schematic of chicken viperin mapped to chicken chromosome 3 (Accession Number: NW 00147161.1) between CMPK2 and RNF144A genes, displaying a syntenic relationship with the humans locus, as indicated. Genes are transcribed in the direction of the arrows. (B) Chicken viperin contains 6 coding exons.

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according to manufacturer’s instructions (Applied Biosystem). Primers and probes (Table 1), where possible, were designed across intron/exon boundaries and labelled with the reporter dye carboxyfluorescein (FAM) and the quencher tetramethyl6-carboxyrhodamine (TAMRA). PCR cycling was performed as follows: 95 ◦ C for 15 s, 61 ◦ C for 30 s and 68 ◦ C for 30 s. Error was expressed as the standard error of the mean (SEM). 3. Results 3.1. Identification of the chicken ISG viperin Viperin specific primers were designed based on the predicted chicken viperin sequence (XM 426208) and are presented schematically (Fig. 1A). PCR-amplification using the primers vip1R and vip1F combined with cDNA template (derived from chicken splenocytes treated with IFN-␣) yielded an 818 bp product corresponding to the 3 end of the viperin CDS (Fig. 1B). A similar strategy failed to amplify the 5 end of the viperin CDS, but a product was derived using 5 RACE with the primers vipOUT and 5 RACE outer (Fig. 1C). This was followed by nested PCR using vipIN1 or vipIN2 primers and the outer 5 RACE (Fig. 1D). These products matched the expected size of chicken viperin fragments (Fig. 1A–D) and were confirmed by sequencing then assembled and deposited into GenBank (Accession Number EU427332). The obtained sequence (Fig. 2) of 1065 bp, encoding 354 amino acids matched the in silico predicted sequence. The viperin gene mapped to chicken chromosome 3 and was flanked by the 2 genes, CMPK2 and RNF144A, showing syntenic similarity to the human locus on chromosome 2 (Fig. 3A). The chicken viperin sequence is composed of 6 exons (Fig. 3B), similar to the orthologues of other species. 3.2. Chicken viperin is structurally similar to mammalian orthologues A comparison of chicken viperin to orthologous genes in other animals showed a minimum of 71% sequence identity (Table 1). Chicken and turkey viperin sequence was 96% identical and had 99% overall sequence similarity (Table 1). The zebra finch displayed high sequence identity (92%), despite this predicted viperin orthologue was approximately 63 amino acids shorter compared to other viperin sequences (Table 1). Outside the class of aves, both the Chinese-soft-shell-turtle and cow had high sequence identity at 76% and overall total similarity of 90% and 88% respectively (Table 1), consistent with their evolutionary relationships. The lowest sequence identity was found between the chicken and zebrafish at 71% (Table 1). Multiple sequence alignments of the investigated species showed that the viperin sequences were highly conserved overall, however, with the exception of the aves there was high dissimilarity within the N-terminal 77 amino acids (Fig. 4). Within the variable N-terminal region of chicken viperin a leucine zipper, a heptad repeat of five leucines, was identified and appears to be conserved in all species (Fig. 4). In addition, 4 characteristic radical S-adenosyl methionine (SAM) motifs were identified in the chicken sequence. The first includes an iron–sulphur motif CxxxCxxC followed by conserved motifs found in other radical SAM enzymes (Fig. 4). The various viperin sequences were used to create a phylogenetic tree; unsurprisingly, chicken viperin clustered with its nearest relatives the turkey and zebra finch (Fig. 5). 3.3. Chicken viperin is upregulated in vitro following treatment with various TLR ligands In mammals, viperin is rapidly induced by several pathogenrelated signals independent of IFN signalling. Viperin was

Fig. 4. Multiple alignment of chicken viperin with orthologues from other animals. The chicken viperin amino acid sequence was aligned with viperin from other animals using the CLUSTALW program and revealed identical (*), conserved (:), and semi-conserved (.) residues of viperin. The N-terminal leucine zipper residues are in bold, potential leucine zipper is circled, and the conserved viperin motifs are boxed and numbered I–IV.

upregulated in the chicken in response to TLR ligands following treatment of chicken splenocytes with the TLR3 ligand, poly(I:C) (Fig. 6A). The viperin expression was detected at 2 h post-treatment, and increased to 112-fold at 4 h and 110-fold at

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Fig. 5. Phylogenetic analysis of the viperin genes from a range of animals. A rooted tree was constructed from the amino acid sequences of viperin genes using an unweighted pair group method with arithmetic mean using the ClustalW program. GenBank accession numbers of the respective genes can be found in Table 1.

6 h relative to unstimulated controls (Fig. 6). When the poly(I:C) was transfected into the chicken splenocytes (targeting the MDA-5 pathway) there was a similarly high induction of viperin expression with 153-fold increase at 4 h and 186-fold increase at 8 h posttreatment (Fig. 6B). Stimulation of chicken splenocytes with LPS (targeting the TLR4 pathway) lead to a slow but steady increase in viperin expression which peaked at 24 h (28-fold increase) followed by viperin decline at 48 h (Fig. 6C). In contrast, no significant changes occurred following cell treatment with endotoxin-free bacterial DNA over a 24 h period (Fig. 6D). Similarly, treatment with Loxoribine (targeting the TLR7 pathway) did not lead to any significant increase in viperin expression – and appeared to downregulate at 2 and 6 h (Fig. 6E). Since, ISGs are typically activated by IFN, chicken splenocytes were treated with 1 ␮g of IFN-␣ up to 24 h. Viperin expression peaked at 2 h with a 29-fold increase and was markedly increased over Mx, which showed a peak level of 16-fold at 4 h relative to controls (Fig. 6F). 3.4. Chicken viperin is upregulated during viral infections In mammals, viperin is upregulated during several virus infections, including influenza (Hinson and Cresswell, 2009; Wang et al., 2007). With this in mind chicken viperin levels were tested in the lung and spleen following experimental infection with H5N1 (A/Muscovy duck/Vietnam/453/2004) avian influenza virus (Burggraaf et al., 2014). The lung of infected chickens displayed a 24-fold increase in viperin levels whilst Mx and PKR increased 35fold and 10-fold respectively (Fig. 7A). In the spleen viperin was upregulated 169-fold, while Mx and PKR upregulated 119-fold and 12-fold respectively, relative to the uninfected controls (Fig. 7B). Viperin levels were also tested in chickens experimentally infected with a classical Australian IBDV strain (01/09) and viperin was upregulated 198-fold whilst Mx upregulated 506-fold in the bursa (Fig. 7C). A similar trend was observed in chickens infected with a variant Australian IBDV strain (21/06, S. Sapats, CSIRO) where viperin and Mx levels increased in the bursa 77-fold and 280-fold, respectively (Fig. 7C). In the spleen the level of viperin and Mx was generally lower following infection with classical IBDV with 11-fold and 61-fold increases in viperin and Mx, respectively (Fig. 7D). Similar trends were evident following infection with the variant IBDV strain where viperin and Mx levels increased by 20-fold increase and 113-fold relative to the uninfected controls (Fig. 7D). 4. Discussion Viperin is a highly conserved ISG, belonging to a super-family of enzymes known as radical SAM, and contributes to the early innate immune response against a variety of viral and bacterial infections in mammals (Fitzgerald, 2011; Jiang et al., 2008). Here we describe the viperin orthologue in the chicken in an effort to

expand knowledge of the IFN pathway and ISGs in the chicken. Bioinformatics and PCR show the chicken viperin gene has a predicted translation of 354 amino acids – a protein that was slightly smaller than viperin in other species with the exception of the zebrafish. The chicken viperin sequence maps to chromosome 3 and its location between the genes, CMPK2 and RNF144A, is a similar arrangement to the human viperin locus on chromosome 2, highly supporting its viperin designation. Chicken, mammalian and fish viperin amino acid sequences appear to be highly conserved with at least 70% identity. Three key domains previously identified in mammalian viperin can be identified in chicken viperin. The N-terminal variable region spanning 77 amino acids; a central radical SAM domain and a C-terminal conserved region (Boudinot et al., 1999; Jiang et al., 2008). Furthermore, chicken viperin contains 5 leucine residues and 4 of these align with leucine zippers in viperin from other species. Leucine zippers typically contain 4–5 leucine residues at every 7th amino acid position, which facilitate protein–protein interactions with other leucine zippers (Landschulz et al., 1988). Functional studies confirm the reduced antiviral effects of mammalian viperin following leucine zipper to alanine mutations (Jiang et al., 2008). In addition, viperin typically possesses radical SAM signatures, that include three closely spaced cysteine residues in a characteristic CxxxCxxC motif (Boudinot et al., 1999; Jiang et al., 2008). There are three conserved radical SAM motifs present in chicken viperin; molybdopterin (motif II), heme D1 (motif III) and PQQ (motif IV) (Boudinot et al., 1999; Jiang et al., 2008). In mammals, mutation of viperin radical SAM motifs show their importance for antiviral activity against West Nile virus (WNV) and Dengue virus infection (Jiang et al., 2008, 2010). Collectively, the evolutionary conservation and the preservation of key domains in chicken viperin suggests its importance in the innate immune response. In mammals, viperin is a highly inducible ISG and is rapidly increased during viral infections (Proud et al., 2008; Rivieccio et al., 2006; Severa et al., 2006; Wang et al., 2007). Furthermore, viperin is highly up-regulated following treatment of cells with the viral mimetic, poly(I:C). In chickens, viperin was rapidly inducible by poly(I:C) at 2 h and peaked between 4 and 6 h consistent with previous observations in mammals (Rivieccio et al., 2006; Saitoh et al., 2011). Previous virus infection models suggest that viperin is induced via RIG-I, and to a lesser extent MDA5 activation (Severa et al., 2006). Since chickens lack RIG-I but maintain the MDA-5 pathway, it is suggested that viperin expression may be driven by MDA-5 or possibly TLR3 (Barber et al., 2010; Karpala et al., 2011). Accordingly, when poly(I:C) was transfected into cells only one tenth the concentration of poly(I:C) was required to induce viperin to the levels observed following untransfected poly(I:C). Thus, viral replication products such as dsRNA are likely to induce viperin in chickens, and may contribute to host survival.

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A

B

10 μg/mL poly(I:C)

1 μg/mL poly(I:C)-transfected 250

160

viperin

140

Mx

** *

100

*

* *

80 60

Mx

200

relave expression

120

relave expression

* viperin

*

40

**

* *

150

100

** *

50

20

* 0

0

2

4

6

8

0

24

2

0

me post smulaon (h)

D

40

viperin 35

Mx

20 15

* *

*

10

relave expression

relave expression

3

2

1

* *

5 0

3

0

1 mMloxoribine

6

24

0

48

me post smulaon (h)

F

relave expression

3

2

1

0

2

viperin

30

Mx

4

6

24

me post smulaon (h)

1 μg/ml IFN-α 35

viperin Mx

relave expression

viperin

Mx

25

4

24

2 μg/mL EF-DNA 4

*

30

E

8

me post smulaon (h)

10 μg/mL LPS

C

4

***

25 20 15

**

*** ***

10

***

*

** 5

0

0

2

6

24

me post smulaon (h)

0

0

2

4

6

24

me post smulaon (h)

Fig. 6. Stimulation of viperin mRNA by various ligands. Chicken splenocytes were stimulated with 10 ␮g/mL of poly(I:C) (A), transfected with 1 ␮g/mL of poly(I:C) (B), stimulated with 10 ␮g/mL of LPS (C), 2 ␮g/mL of EF-DNA (D), 1 mM of loxoribine (E), 1 ␮g/mL of IFN-␣ (F) over a 48 h period. Cells were analysed for viperin and Mx expression by qRT-PCR. Data represents mean fold expression relative to unstimulated controls. Asterisks indicate statistically significant differences between untreated and other groups, where *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3, error bars = SE).

Additional TLR ligands were tested for their impact on viperin regulation. Bacterial LPS (via TLR4) and endotoxin free bacterial DNA (via TLR21), were cultured with chicken splenocytes and similar to mammalian reports, viperin expression was found to be upregulated. Chicken viperin expression was slowly induced by TLR4, peaking at 24 h, and may be the result of IFN production since others have found that LPS can directly up-regulate IFN-␣ as early as 1 h (Jacobs and Ignarro, 2001). We confirm that in chickens, recombinant IFN-␣ induces viperin expression with mRNA detected at 2 h post-treatment. Similar to what others have observed viperin levels returned to near unstimulated levels by 24 h (Chan et al., 2008). The

early IFN induction of viperin was similar to poly(I:C) induction of viperin and therefore supports that poly(I:C) can directly stimulate viperin. Future tests could employ neutralising antichicken–IFN␣ antibody in poly(I:C) treatments to block IFN activity. Overall the induction of chicken viperin exhibited similar kinetics to Mx. During influenza infection viperin localises to the ER and inhibits the trafficking of soluble virally-encoded proteins thus restricting viral spread (Hinson and Cresswell, 2009; Wang et al., 2007). Mammalian in vitro studies have found that viperin expression reduced virus replication and restricted viral budding (Tan et al.,

K.E. Goossens et al. / Molecular Immunology 63 (2015) 373–380

A (AI-infected lung) 60

50

B

379

(AI-infected spleen)

viperin

250

viperin

Mx

200

Mx

**

PKR

*

PKR 150 100 relave expression

relave expression

40

30

20

16

***

14 12

10

10 8 6

0

4 2 0 Uninfected

D

160

viperin

Mx

140

Mx

PKR

120

PKR

*

100

*

80

3.5 3 2.5 2

Infected

(IBDV-infected spleen)

viperin

relave expression

900 800 700 600 500 400 300 200 100

Uninfected

Infected

C (IBDV-infected bursa)

relave expression

50

60 40 30 25 20 15

1.5 10

1

5

0.5 0 Uninfected

Classical

Variant

0 Uninfected

Classical

Variant

Fig. 7. Induction of chicken viperin and other ISGs during infection. Expression of chicken viperin, Mx and PKR was determined by qRT-PCR analysis of H5N1 avian influenza infected lung (A) and spleen (B) and infections with classical and variant strains of IBDV in the chicken bursa (C) and spleen (D), compared to uninfected controls. Data represents the mean fold expression of the ISG’s in infected samples relative to uninfected controls. Asterisks indicate statistically significant differences between untreated and other groups, where *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3, error bars = SE).

2012). Since avian influenza viruses impact the poultry industry, the role of viperin during influenza infection in chickens is of considerable interest. We found that viperin mRNA levels were up-regulated in the lung and spleen of infected chickens. Viperin restriction of influenza in the chicken however, requires further investigation. The role of ISGs, such as viperin, in IBDV infection is currently unknown. Previous IBDV investigations found increased bursal levels of type II IFN and interleukins; IL-1␤, IL-6, IL-18, IL-4 and IL-13 (Eldaghayes et al., 2006; Palmquist et al., 2006). In vitro microarray studies have found that Mx is up-regulated following IBDV infection (Wong et al., 2007). During in vivo infection with the classical IBDV strain, in which birds show no clinical signs of disease, we found that there was an increase in viperin and Mx mRNA in the bursa – the predominant site of virus replication. In addition, infection with the variant strain of IBDV, in which chickens displayed mild clinical symptoms, the levels of viperin and Mx were similarly up-regulated in the bursa but to a lesser extent than induced by the classical IBDV strain. It

has been suggested that strains of IBDV are capable of modulating the host immune response, in particular, the variant IBDV strains appear to down-regulate the IFN response, although the exact mechanism is unclear (Lukert and Saif, 1997; Eldaghayes et al., 2006). The viperin regulation pattern paired with disease severity associated with the classical and variant IBDV strains support the idea of using IFNs as treatments for viral infection. In summary, chicken viperin is highly conserved and possesses both leucine zipper and radical SAM motifs, which have been shown to be critical to the antiviral actions of viperin in mammals. Chicken viperin was rapidly induced by poly(I:C) or infection with highly pathogenic avian influenza or IBDV. Viperin was also up-regulated in response to LPS suggesting potential antimicrobial actions in addition to its antiviral activities. Furthermore, viperin was upregulated in response to IFN treatment, which is a hallmark of all ISGs. Future work is required to understand role of viperin as an antiviral or antimicrobial in chickens to promote novel therapeutics in the treatment of poultry diseases.

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