Induction of Mx and PKR Failed to Protect Chickens from H5N1 Infection

Abstract

We are currently facing a global threat caused by a highly pathogenic avian H5N1 influenza virus (hpH5N1). Death occurs in 48 h in infected chickens, suggesting that they fail to eliminate the virus. Little is known about the immune response in chickens after hpH5N1 infection, or how the virus is evolving to modify and evade host protective responses. Therefore, to better understand the chicken immune response following hpH5N1 infection, we set up an experimental infection of chickens with an hpH5N1 strain, and quantified the mRNA expression of several cytokines and antiviral proteins at different time points post-infection. We show here that a weak host immune response is observed in vivo, in spite of the induction of IL-6, myxovirus resistance protein (Mx), and protein kinase R (PKR). This weak immune response, probably due in part to the absence of type I interferon, was not sufficient to counteract the hpH5N1 virus and protect the chicken from death.

Introduction

A Highly pathogenic avian influenza virus H5N1 (hpH5N1) continues to circulate in poultry and wild birds, causing considerable concern for veterinary and public health officials in Asia, Europe, and Africa. More importantly, the unusual ability of this virus to infect humans gives rise to growing concerns about the possibility of the virus becoming pandemic in humans (13,16,17).

Infection of chickens with hpH5N1 leads to rapid death within days, thus leaving the innate immune response as the only defense system of relevance for disease resistance. Elucidation of interactions between host and pathogen very early in infection will reveal how the virus is evolving to evade host protective responses, and why the host is unable to control the virus.

While there has been much growth in our understanding of mammalian responses to influenza viruses, the antiviral immune response to avian influenza viruses remains largely unknown in the chicken. Therefore we set up an experimental infection of chickens to collect basic information about the early innate immune response during infection with an hpH5N1 strain (A/duck/hungary/1180/2006, clade 2 H5N1; courtesy of Dr. Vilmos Pálfi).

Materials and Methods

Two groups of 20 6-wk-old Redco layer chickens were housed in separate BSL3 isolators with negative air pressure. Control animals received 100 μL of PBS (50 μL as an eye drop and 50 μL as a nasal cavity drop), and H5N1-infected animals received a virus dose of 10⁵ EID₅₀ diluted in PBS at the same amounts as in the control animals. Four animals from each group were sacrificed at 2, 4, 8, 24, and 30 h post-infection (hpi). The lungs, spleen, cecal tonsils, and duodenum were removed and stored in RNA Later (Qiagen Gmbh, Hilden, Germany), according to the manufacturer's instructions. Sections of 30 mg of tissue were transferred to 600 μL of RLT Lysis Buffer (Qiagen), with DTT added (150 μM final dilution), and homogenized using a Rotor-Stator Homogeniser (IKA Werke Gmbh, Staufen, Germany) for lungs, or a TissueLyser (Qiagen) for other tissues. The resulting lysate was centrifuged for 10 min at 5000 × g and the supernatant was used to prepare RNA using the RNeasy^® Protect Mini Kit (Qiagen), according to the manufacturer's instructions. To avoid contamination with genomic DNA, on a column DNase digestion was performed at 37°C for 15 min (RNase-Free DNase Set; Qiagen). All RNA concentrations were determined using a Nanodrop^® ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE) (Labtech), and all RNA preparations were analyzed using a Lab-on-a-Chip Agilent 2100 Bioanalyzer (RNA 6000 Nano Kit; Agilent Technologies, Santa Clara, CA) to confirm RNA integrity and purity.

Single-stranded complementary DNA (cDNA) was synthesized from 2 μg of total RNA using the ProtoScript^® First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA), according to the manufacturer's instructions. Besides having been treated with DNase, we also checked for DNA contamination of the RNA samples by PCR amplification from cDNA templates using primers overlapping intron 2 of the B-LB II MHC class II gene.

Specific primers were designed for target genes (HA, IL-6, IL-8, type I IFN [α and β], IL-12, IFN-γ, Mx, and PKR) (Table 1), and verified by PCR of genomic DNA and cDNA. Quantitative PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA), using Power SYBR^® Green PCR Master Mix (Applied Biosystems). The cycling conditions were 10 min at 95°C, 40 cycles of 10 sec at 95°C, 30 sec at 60°C, and 30 sec at 72°C, followed by a final step of melting temperature increases up to 95°C.

Table 1.

Primers Used in the Study

	Sense primer	Antisense primer	Fragment length (bp) and Genbank accession no.
Gene	Sequence (5′ → 3′)	Sequence (5′ → 3′)	Genomic DNA (or RNA for HA)		Complementary DNA
Maternal G10 transcript	AGC CTC ATG AAG GGA AGA GGA	CAG GCA GCA GAG GTT CTC ATA	442	NC006101	206	XM001233097
GAPDH	GGC AGA TGC AGG TGC TGA GTA T	CGT CTT CTG TGT GGC TGT GAT G	737	NM204305	131	XM001235464
β-actin	GTC CAC CGC AAA TGC TTC TAA A	CCA TGC CAA TCT CGT CTT GTT T	102	NM205518	102	L08165
B-LB II MHC class II	GCC CGT CAC GTA GCA CGC CAG ACG GTC	GAG TGC CAC TAC CTG AAC GGC ACC GAG CGG	410	M29763	325	EF554716
Hemagglutinin	CAA ACT CCA ATG GGG GCG ATA	TTG AGG GCT ATT TCT GAG CCC	280	EU930900	280	EU930900
IFN-α	GAC ATC CTT CAG CAT CTC TTC A	AGG CGC TGT AAT CGT TGT CT	238	AB021154	238	EU367971
IFN-β	CAC AGC CTC CTC AAC CAG AT	CAT GGT CCC AGG TAC AAG CA	181	X92479	181	NM001024836
IL-6	GGA GAA ATG CCT GAC GAA GCT	CTG ACT TCA GAT TGG CGA GGA	1577	AJ250838	221	EU170468
IL-8	GGC TTG CTA GGG GAA ATG A	AGC TGA CTC TGA CTA GGA AAC TGT	200	AJ009800	200	NM205498
IL-12 p40	AGA CTC CAA TGG GCA AAT GA	CTC TTC GGC AAA TGG ACA GT	2235	AJ564202	274	NM213571
IFN-γ	AGC TGA CGG TGG ACC TAT TAT T	GGC TTT GCG CTG GAT TC	1257	Y07922	258	NM205149
Lymphotactin	GGA TTT AAG GGT GAA CAG TAG ATG	TAG AAA TAG AAA GCC CGA GGA T	N.D.	N.D.	256	NM205046
Mx	ATC CAT GGT CCA ACT TCA GC	GCC TCT TGG ACA CTT TCT GC	201	DQ788616	201	EU348752
PKR	CCT CTG CTG GCC TTA CTG TCA	AAG AGA GGC AGA AGG AAT AAT TTG CC	151	AB125660	151	AB125660

Relative quantification by RT-PCR provides accurate comparison between the initial levels of cDNA in each sample, but requires an endogenous control to correct for differences in RNA input. Thus expression changes of GAPDH, β-actin, and the maternal G10 transcript (a homolog to yeast BUD31 gene) were investigated with absolute RT-PCR of two animals from each group (infected and noninfected at 2, 4, 8, 24, and 30 hpi). The maternal G10 transcript RNA was chosen as an endogenous control because it had the lowest standard deviation calculated between all the different conditions, and therefore it was used as an endogenous control in relative quantification assays to determine the viral load and the cytokine responses (for the viral load, direct CT comparison gave the same results; data not shown).

Results were analyzed using 7300 System Sequence Detection Software v1.4 and RQ Study Application (Applied Biosystems), and are presented as log₂ (mean ddCT infected/mean ddCT noninfected), with ddCT being the fold change (relative quantity) of target mRNA, normalized to maternal G10 transcript mRNA and relative to a calibrator sample.

Immunostaining was performed using 4% paraformaldehyde fixation, with anti-NP monoclonal antibody HB65 as the first antibody (ATCC), and anti-mouse HRP-coupled antibody (Dako Group, Glostrup, Denmark) or anti-mouse FITC-coupled antibody (Sigma-Aldrich Co., St. Louis) as the secondary antibody.

Results

We investigated the presence of viral RNA by measuring the level of the viral hemagglutinin (HA) RNA, to gain insight into the tropism and dissemination of the virus. Quantitative measurements of viral RNA in lung, spleen, cecal tonsil, and duodenum at 24 and 30 hpi revealed much higher levels in lung and spleen at both time points, although all the tested tissues were infected at 24 h (Fig. 1A). Furthermore, virus was detected in lung and cecal tonsils as early as 12–24 hpi, using immunofluorescence or HRP staining of 5-wk-old SPF birds infected with 10⁶ EID₅₀ (Fig. 1B–E).

FIG. 1.

Detection of hpH5N1 after infection by quantitative RT-PCR and immunohistological analysis. (A) Quantitative RT-PCR analysis of viral hemagglutinin RNA in lung, spleen, cecal tonsils, and duodenum. (B and D) Immunofluorescence labeling of viral NP protein in lung and cecal tonsils, respectively. (C and E) Immunoperoxidase labeling of viral NP protein in lung and cecal tonsils, respectively.

Interestingly, we also detected the viral RNA at 24 hpi in the thymus (data not shown), suggesting that perhaps subsets of T cells may support replication of hpH5N1 in vivo. However, this must be confirmed by immunohistochemistry.

Elevated IL-6 expression mediated by TLR4 and NF-κB signaling was shown to be associated with acute lung injury in mice infected with H5N1 virus (9). We detected an increase in IL-6 RNA at 24 hpi in all the infected tissues, but no significant induction of IL-8, even at 30 hpi (Fig. 2A and B). It was reported that in ferrets exhibiting severe disease after influenza infection, IL-6 was induced, but no induction of IL-8 was detected (22). It was also demonstrated that a reconstructed 1918 influenza virus was a good IL-6 inducer in macaques, but it did not significantly induce IL-8 (10).

FIG. 2.

Quantitative RT-PCR analysis of cytokine, Mx and PKR gene expression at 2, 4, 8, 24, 30 hpi by hpH5N1 in lung, spleen, caecal tonsils, and duodenum: (A), IL-8 (B), IL-12 (C), IFN-γ (D), IFN-α (E), IFN-β (F), Mx (G), and PKR (H) genes.

Although type I interferons play a critical role in resistance to virus infection and induction of adaptative immunity effector responses, we did not observe any significant induction of IFN-α and IFN-β RNA, even at 30 hpi (Fig. 2E and F). In order to confirm our measurement system, we measured IFN-α RNA in infectious bursal disease virus (IBDV)-infected samples. IBDV is a small RNA virus (family Birnaviridae) that specifically targets early B cells, especially those in the bursa of Fabricius (Fig. 2E). This provides a model of an acute local infection with innate response and cytokine induction (19). Indeed, we noted that contrary to H5N1-infected samples, we found strong induction of IFN-α RNA in the bursa after IBDV infection (several hundred times).

These results are consistent with reports indicating inhibition of IFN responses after H5N1 infection. Notably, IFN-β induction is attenuated in polarized human epithelial cells infected with H5N1 virus (24). Moreover, it was reported that this inhibition of type I interferon transcription is indirectly due to the NS1 protein of highly pathogenic influenza A virus (3,4,6,11,12,14,15,20,26).

Unlike IFN-α/β, which are immediately induced in response to invading pathogens, several cytokines are known to stimulate the production of IFN-γ, including IL-12. No induction of IL-12 or IFN-γ RNA was detected, even at 30 hpi in any of the tissues (Fig. 2C and D). This result was corroborated by the absence of lymphotactin RNA induction (data not shown), which would be expected from IFN-γ-driven polarization of naïve T cells toward Th-1 cells.

Since the antiviral innate immunity is not restricted to IFN effects, we measured expression of the Mx (myxovirus resistance protein) and PKR (protein kinase R) genes in the noninfected and infected tissues. These proteins are involved in the response of cells to viral infections. We observed an increase in Mx mRNA expression in all infected tissues, from two- to fivefold at 24 hpi, and from three- to 10-fold at 30 hpi, depending on the tissue (Fig. 2G). The effect was largest in the lung, where we also observed increased PKR mRNA expression (Fig. 2H).

Discussion

Mx, induced principally by type I IFN, is one of the most important antiviral proteins, as it inhibits different steps of the influenza virus replication cycle (18). This increase in the expression level of Mx in the absence of type I IFN could be explained by an alternative pathway, in which no new synthesis of IFN protein is needed. This has been observed in several species, including salmon (1) and mouse (8), but until now to our knowledge, not in birds. This mechanism seems to be conserved throughout several species. Indeed, promoter analysis of Mx genes in several species showed that it contained several other motifs than the interferon-stimulated response element (5,8,23). In chickens, the Mx promoter (accession no. DQ788615) contains putative binding motifs for NF-κB and SP1 (5,23). In addition, we identified an IL-6 type II-responsive element (CTGGGA) (5,7,25), and an IL-6 type I-responsive element (also called the NF-IL-6 responsive element, or CAAT/enhancer binding protein-β [C/EBP-β]) (2), at positions −142 and −79, respectively.

These results suggest that the induction of the antiviral proteins PKR and Mx seen at 24 hpi fails to produce an effective antiviral response against hpH5N1 AI virus. The induction of antiviral proteins such as Mx, PKR, and ISG20 (data not shown) was insufficient to limit viral replication. In this regard, it is noteworthy that upregulation of the Mx1 gene has been shown to increase after infections with influenza viruses in mammals, and the Mx proteins have been generally found to block the function of essential viral proteins. However, it was reported that Mx protein may not have antiviral activity in any chicken breed (21).

Conclusion

In summary, this strain of hpH5N1 appears to suppress or delay the expression of critical host genes involved in the innate immune response (IFN-α/β and IL-8) very early in infection. This may be one of the mechanisms that gives this virus a survival advantage. Even after infection, the virus may evade the innate immune system by multiplying extremely quickly, and thus renders the antiviral host proteins ineffective.

Footnotes

Acknowledgments

This research was supported by funds from the European Commission (SSPE-CT-2006-44372 Inn-Flu). We thank Dr. M.K. Chelbi-Alix and Dr. J. Young for helpful input.

Author Disclosure Statement

No conflicting financial interests exist.

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