Downloaded from www.microbiologyresearch.org by IP: 54.159.222.200 On: Tue, 17 May 2016 18:45:23 Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses Aleksandr S. Lipatov, 1 Samita Andreansky, 2 3 Richard J. Webby, 1 Diane J. Hulse, 1 Jerold E. Rehg, 3 Scott Krauss, 1 Daniel R. Perez, 1 4 Peter C. Doherty, 2,5 Robert G. Webster 1,4 and Mark Y. Sangster 2 1 Correspondence Robert G. Webster robert.webster@stjude.org 1,2,3 Departments of Infectious Diseases (Division of Virology) 1 , Immunology 2 and Pathology 3 , St Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105–2794, USA 4 Department of Pathology, University of Tennessee, Memphis, TN 38163, USA 5 Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia Received 5 October 2004 Accepted 10 December 2004 The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu 92 RAsp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50 %) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1a, IL1b, IL6, IFN-c and chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease. INTRODUCTION In 1997 in Hong Kong, avian H5N1 influenza A virus (H5N1/97) was transmitted directly from poultry to humans with an overall case-fatality rate of 33 % (Claas et al., 1998; Subbarao et al., 1998). The severity and high mortality rate were not explained by underlying disorders (Yuen et al., 1998). Affected patients had a primary viral pneumonia complicated by acute respiratory distress, multiple organ dysfunction and haemophagocytosis (To et al., 2001; Yuen et al., 1998), all of which are associated with cytokine dysregulation (Fisman, 2000; Headley et al., 1997). Despite numerous studies in animal and cell models, the basis of the unusual pathogenicity of Hong Kong H5N1/97 influenza viruses remains unclear. In the lungs and spleens of infected mice, all of the H5N1/ 97 viruses isolated from humans induce a high con- centration of cytokines, such as interleukin (IL)-1b and interferon (IFN)-c, and of the chemokine macrophage inflammatory protein (MIP)-1a, (Tumpey et al., 2000). In human primary monocyte-derived macrophages, the H5N1/97 viruses induced much higher transcription of pro-inflammatory cytokines, particularly tumour necrosis factor (TNF)-a and IFN-b, than did H3N2 or H1N1 viruses 3Present address: Department of Pediatrics, Steele Memorial Children’s Research Center, University of Arizona, Tucson, AZ, USA. 4Present address: Department of Veterinary Medicine, University of Maryland, 8075 Greenmead Drive, College Park, MD 20742-3711, USA. 1Present address: Department of Microbiology, The University of Tennessee, Walters Life Sciences F419, 1414 W. Cumberland Avenue, Knoxville, TN 37996, USA. 0008-0663 G 2005 SGM Printed in Great Britain 1121 Journal of General Virology (2005), 86, 1121–1130 DOI 10.1099/vir.0.80663-0