Virus Research 163 (2012) 151–159 Contents lists available at SciVerse ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Human intestinal epithelial cells are susceptible to influenza virus subtype H9N2 Bingqian Qu a , Xue Li a , Wei Gao a , Wenkui Sun c , Yu Jin a,d , Carol J. Cardona b , Zheng Xing a,b,∗ a The Key Laboratory of Pharmaceutical Biotechnology and Medical School, Nanjing University, Nanjing, China b Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Twin Cities, St. Paul, MN, USA c Medical School of Second Military Medical University, Shanghai, China d Nanjing Children’s Hospital, Nanjing, China article info Article history: Received 26 June 2011 Received in revised form 2 September 2011 Accepted 4 September 2011 Available online 22 September 2011 Keywords: Avian influenza virus H9N2 Intestinal epithelial cells Apoptosis Innate immune response abstract Avian influenza viruses (AIV) replicate efficiently in guts of birds, and virus shedding is critical to viral transmission among birds and from birds to other species. In this study, we showed that an H9N2 viral strain, isolated from a human patient, caused typical influenza-like signs and illness including loss of body weight in Balb/c mice, and that viral RNA could be detected in intestinal tissues. We demonstrated that human intestinal epithelial cell line HT-29 was susceptible to the virus, and the infected cells went apoptotic at the early stage post infection. Compared to a pandemic (H1N1) 2009 influenza isolate, we found that the human H9N2 virus induced more severe apoptotic and stronger innate immune responses. Both extrinsic and intrinsic apoptotic pathways were activated in human intestinal epithelial cells, and the levels of FasL and TNF- were induced up to hundreds-fold in response to the H9N2 infection. Inter- estingly, Bcl-2 family member Bid was cleaved during the course of infection, and the truncated Bid (tBid) appeared to play a role in the initiation of the intrinsic apoptosis with increased release of cytochrome c in cytosol. As for pro-inflammatory responses in H9N2-infected intestinal epithelial cells, RANTES and IP10 were induced significantly and may have played a major role in intestinal pathogenicity. More- over, TLR-8, MyD88, and MDA-5 were all up-regulated in the infection, critical in the induction of IFN- and host innate immunity against the H9N2 virus. Our findings have demonstrated a unique pattern of host responses in human gut in response to H9N2 subtype influenza viruses, which will broaden our understanding of the pathogenesis of AIV infection in both humans and animals. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Influenza A viruses are single-stranded RNA viruses belong- ing to the Orthomyxoviridae family. Although all 16 subtypes of influenza A viruses have been discovered in waterfowl, there are only three subtypes of human influenza viruses which include H1, H2, and H3, and some have caused periodic epidemics or pan- demics (Horimoto and Kawaoka, 2005; Kim et al., 2009). Among avian influenza viruses (AIV), three subtypes including H5, H7, and H9 can infect humans and some of H5 and H7 subtypes are highly pathogenic. The outbreak of human infection with AIV H5N1, iden- tified in Hong Kong in 1997, caused a severe respiratory syndrome in 18 people, of which one-third died (Claas et al., 1998; Yuen et al., 1998). To date, more than 500 human infections with this virus have been reported by the World Health Organization (WHO) world- wide, with a mortality rate of up to 60%. H9N2 influenza viruses have been circulating in poultry in Asia and North America for ∗ Corresponding author at: 301F Veterinary Science Building, 1971 Common- wealth Avenue, Saint Paul, MN 55108, USA. Tel.: +1 612 626 5392. E-mail address: zxing@umn.edu (Z. Xing). decades, and have evolved and successfully crossed the species bar- rier to infect humans (Lin et al., 2000) since the late 1990s. In 1999 and 2003, H9N2 influenza viruses were isolated from the nasopha- ryngeal aspirate of several children with an influenza-like illness in Southern China and Hong Kong (Butt et al., 2005; Peiris et al., 1999). It is noteworthy that six genomic segments of the H9N2 viruses encoding the internal proteins were related to the high pathogenic- ity H5N1 viruses isolated from birds and patients (Guan et al., 2000), indicating the importance of H9N2 viruses in the genesis of deadly viruses which may have pandemic potential. Most AIV subtypes cause asymptomatic infection in waterfowl and poultry, but a few are highly pathogenic even in waterfowl (Kim et al., 2009). Despite their different pathogenicity, both highly and lower pathogenic AIV replicate in avian guts and spread by fecal-oral transmission (Webster et al., 1978). Amino acids in hemagglutinin (HA) residues 226 and 228 and the type of sialic acid receptors on the target cells are critical for the virus to infect duck intestine (Ito et al., 2000; Vines et al., 1998). Many human patients infected with AIV H5N1 or pandemic (H1N1) 2009 suffered from relatively high rates of gastrointestinal (GI) tract symptoms (Louie et al., 2009; Uiprasertkul et al., 2005). It was only recently that an autopsy sample from an H5N1-infected patient indicated the 0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.09.007