Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses

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Abstract

In order to understand the molecular mechanisms by which different strains of avian influenza viruses overcome host response in birds, we used a complete chicken genome microarray to compare early gene expression levels in chicken embryo fibroblasts (CEF) infected with two avian influenza viruses (AIV), A/CK/Hong Kong/220/97 and A/Egret/Hong Kong/757.2/02, with different replication characteristics. Gene ontology revealed that the genes with altered expression are involved in many vital functional classes including protein metabolism, translation, transcription, host defense/immune response, ubiquitination and the cell cycle. Among the immune-related genes, MEK2, MHC class I, PDCD10 and Bcl-3 were selected for further expression analysis at 24 hpi using semi-quantitive RT-PCR. Infection of CEF with A/Egret/Hong Kong/757.2/02 resulted in a marked repression of MEK2 and MHC class I gene expression levels. Infection of CEF with A/CK/Hong Kong/220/97 induced an increase of MEK2 and a decrease in PDCD10 and Bcl-3 expression levels. The expression levels of alpha interferon (IFN-α), myxovirus resistance 1 (Mx1) and interleukin-8 (IL-8) were also analyzed at 24 hpi, showing higher expression levels of all of these genes after infection with A/CK/Hong Kong/220/97 compared to A/Egret/Hong Kong/757.2/02. In addition, comparison of the NS1 sequences of the viruses revealed amino acid differences that may explain in part the differences in IFN-α expression observed. Microarray gene expression analysis has proven to be a useful tool on providing important insights into how different AIVs affect host gene expression and how AIVs may use different strategies to evade host response and replicate in host cells.

Introduction

Infection of poultry with highly pathogenic avian influenza viruses (AIV) is associated with systemic infection and high mortality (de Jong and Hien, 2006). From the pathophysiological point of view, the mechanisms that are responsible for severe illness and death with highly pathogenic AIV can vary (Swayne, 2007). In contrast to mammals, there is limited information concerning the molecular pathogenesis of AIV and the regulation of host genes response after AIV infection in avian species.

Previous reports have pointed towards the importance of apoptosis as a significant contributor to the pathogenesis of AIV. Induction of apoptosis by AIV has been shown in chickens (Ito et al., 2002, Van Campen et al., 1989) and in an avian lymphocyte cell line (Hinshaw et al., 1994, Van Campen et al., 1989). In addition, chickens infected with a highly pathogenic avian influenza strain exhibited an increase in serum transforming growth factor-β (TGF-β) activity (Suarez and Schultz-Cherry, 2000), which has been associated with influenza A virus-induced apoptosis (Schultz-Cherry and Hinshaw, 1996).

Cytokine responses to influenza A viruses are central to influenza pathogenesis in mammals (de Jong and Hien, 2006). In avian cells, a recent study showed that IFN-stimulated genes (ISG12, LY6E and haemopoiesis related membrane protein 1 gene) were up-regulated following infection with a highly pathogenic AIV (Zhang et al., 2007). However, AIVs have also been shown to antagonize IFN-α/β production in chicken embryo fibroblasts (CEF) and in chickens (Cauthen et al., 2007, Li et al., 2006). Furthermore, microarray analysis of monocytes/macrophages infected with low pathogenic AIV showed repression of interferon receptor gene expression (Keeler et al., 2007). The ability of AIV's to antagonize IFN activity has been linked to the viral NS1 protein (Cauthen et al., 2007, Li et al., 2006). Moreover, Li et al. (2006) showed that amino acid residue Ala149 of the NS1 protein correlates with the ability of avian influenza viruses to antagonize IFN induction in CEFs.

Other immune response elements, such as the Mx protein, have been shown to play a role in influenza virus infections (Krug et al., 1985, Pavlovic et al., 1992, Ruff, 1983, Staeheli and Haller, 1987). However, the contribution of avian Mx proteins as antiviral elements in AIV infection in birds is not well defined. Transfected chicken cells expressing chicken Mx protein showed no enhanced resistance to several viruses including influenza A virus (Bernasconi et al., 1995). It also has been reported that Mx variations occur in different breeds of chicken; however, in some breeds the Mx gene demonstrated to confer positive antiviral responses to influenza virus while others did not (Ko et al., 2002).

To better understand differences in host responses to infection with distinct AIVs, we initially compared levels of gene expression in CEFs infected with AIVs using microarray technology. Microarray-based gene expression profiling technology has proven to be a powerful tool that helps in the identification of several thousands of transcripts and compares the expression patterns between many different samples (Lockhart et al., 1996). It has been used successfully in the investigation of virus–host interactions in many model systems (Almeida et al., 2007, Geiss et al., 2002, Keeler et al., 2007, Li et al., 2008, Mo et al., 2007, Munir et al., 2005, Tong et al., 2004). Over the past few years several chicken microarrays have been developed (Afrakhte and Schultheiss, 2004, Burnside et al., 2005, Li et al., 2008, Smith et al., 2006, van Hemert et al., 2003). In this study we chose a 60-mer 44K chicken whole genome custom array. This microarray has been previously characterized using different chicken tissues and primary chicken trachea epithelial cells, and has proven to be useful to investigate different biological processes (Li et al., 2008, Zaffuto et al., 2008). In addition to the microarray studies, semi-quantitive RT-PCR was performed on specific genes involved in the innate immune response to study their expression later in the course of infection. The results of these studies can greatly enhance our understanding of the molecular mechanisms related to AIV infection.

Section snippets

Viruses, cells and antibodies

Two highly pathogenic avian influenza H5N1 viruses, A/CK/Hong Kong/220/97 (CK/HK/97) and A/Egret/Hong Kong/757.2/02 (Egret/HK/02), were used in this study. Both viruses produce 100% mortality in chickens after intranasal inoculation; however, CK/HK/97 induces a lower mean death time and replicates to higher titers in tissues than Egret/HK02 (Pantin-Jackwood-unpublished data). Virus stocks were prepared from the second passage in 10-day-old embryonated chicken eggs. The allantoic fluid collected

Growth kinetics of H5N1 viruses

Comparison of the growth characteristics of CK/HK/97 and Egret/HK/02 in CEF are shown in Fig. 1. CEFs support growth of both viruses with titers increasing until 48 hpi. There were no significant differences in virus titers up to 12 hpi, however at 24, 36, 48, and 56 hpi, statistical differences were observed. The titer of CK/HK/97 was 1000-fold higher at 48 hpi relative to Egret/HK/02. Differences in replication of these two viruses in CEF are consistent with their relative phenotypes in chickens.

Gene expression profile of Egret/HK/02 versus CK/HK/97 infected CEFs

Discussion

Very early in the infection, viruses are capable of triggering a series of intracellular events which may be accompanied by changes in host gene expression (Iannello et al., 2006, Korth and Katze, 2002). Influenza viruses in particular have acquired the capability to take advantage and, in many cases, interfere with a wide range of antiviral cellular immune responses to efficiently replicate and propagate (Battcock et al., 2006, Garcia-Sastre, 2001, Hayman et al., 2006, Hinshaw et al., 1994,

Conclusion

Microarray gene expression analysis has proven to be a useful tool for providing clues to the mechanisms involved in pathogenicity of AIVs. While future studies are necessary to better characterize how AIVs differentially affect host response, this study is the first step in understanding the complex events that occur during interactions between cells and AIVs. This study demonstrated that AIVs have varied ability to regulate host genes and this regulation initiates very early in the course of

Acknowledgements

The authors wish to thank Diane Smith, Tracy Smith-Faulkner, and Kristin Zaffuto for their technical assistance; Melissa Scott and Joyce Bennett at the SAA sequencing facility at SEPRL for sequencing of host genes. The authors also would like to thank Mark Jackwood and Aleksandr Lipatov for reviewing this manuscript.

This research was supported by USDA/ARS CRIS project # 6612-32000-039. Mention of trade names or commercial products in this manuscript is solely for the purpose of providing

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