Influenza A viruses cause recurrent outbreaks of local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On June 11, 2009, the WHO declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses1. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) appear to be mild; however, more than 50% of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To better assess the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in nonhuman primates, causes more severe pathologic lesions in the lungs of infected mice, ferrets, and nonhuman primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting these compounds as a first line of defence against the recently declared S-OIV pandemic.

The life cycle of calicivirus is not fully understood because most of the viruses cannot be propagated in tissue culture cells. We studied the mechanism of calicivirus entry into cells using feline calicivirus (FCV) The family Caliciviridae comprises a diversity of pathogens in humans and animals. Studies of the life cycle of the virus have been quite limited because of the lack of cell culture methods for most caliciviruses (13). Porcine enteric calicivirus in the genus Sapovirus is known to propagate in vitro with the addition of an intestinal content fluid filtrate, and Chang et al. (6) recently identified bile acids as molecules responsible for culture in cells. Studies with virus-like particles showed that ABH histo-blood group antigens are ligands of noroviruses belonging to the genus Norovirus (24). Studies in vivo also showed that these antigens are critical for human susceptibility to norovirus infection (17,22). Because noroviruses are not cultivable in cell culture (9), it has not been established whether ABH histo-blood antigens function as receptors for them.,Feline calicivirus (FCV) is a member of the genus Vesivirus and causes upper respiratory tract disease and acute mouth ulceration, occasionally associated with chronic stomatitis and acute arthritis in cats (15,21). It is comparatively easy to study the life cycle of FCV, because the virus replicates efficiently in cell culture without specific supplementation. Kreutz et al. (20) investigated the binding of FCV to feline and nonfeline cells.They reported that FCV bound to feline cells but also showed poor binding ability to nonfeline cells. It was also demonstrated that when nonfeline cells were transfected with genomic RNA of FCV, infectious virus could be recovered from the cells (20). These data suggest that FCV host specificity is restricted in the early stage of FCV infection in cells.In this study, to elucidate the cellular determinant(s) of FCV host specificity, we applied an improved expression cloning method (28,29) to identify cell surface molecules that interact with FCV. As a result, feline junctional adhesion molecule 1 (JAM-1) was identified as a binding receptor for FCV. JAM-1 is a member of the immunoglobulin (Ig) superfamily expressed by various cells and is involved in regulation of cell-cell interactions in the immune system and apical tight junction formation (10, 23). We here demonstrate that feline JAM-1 (fJAM-1) possesses the characteristics required of a functional receptor for FCV. MATERIALS AND METHODS Cells.Crandell-Rees feline kidney (CRFK) cells and human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (FCS). Nonpermissive hamster lung (HmLu-1) cells and African green monkey kidney (Vero) cells were cultured in DMEM with 5% FCS. Murine myeloma P3X63Ag8U.1 (P3U1) cells were grown in RPMI 1640 medium (Sigma) with 10% FCS. Plat-E cells, a 293T-derived murine leukemia virus-based packaging cell line (25), were kindly pr...

h Viral pneumonia has been frequently reported during early stages of influenza virus pandemics and in many human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection. To better understand the pathogenesis of this disease, we produced nonlethal viral pneumonia in rhesus macaques by using an HPAI H5N1 virus (A/Anhui/2/2005; referred to as Anhui/2). Infected macaques were monitored for 14 days, and tissue samples were collected at 6 time points for virologic, histopathologic, and transcriptomic analyses. Anhui/2 efficiently replicated in the lung from 12 h to 3 days postinfection (p.i.) and caused temporal but severe pneumonia that began to resolve by day 14. Lung transcriptional changes were first observed at 6 h, and increased expression of vascular permeability regulators and neutrophil chemoattractants correlated with increased serum leakage and neutrophil infiltration in situ. Additional inflammatory, antiviral, and apoptotic genes were upregulated from 12 h, concurrent with viral antigen detection and increasing immune cell populations. A shift toward upregulation of acquired immunity was apparent after day 6. Expression levels of established immune cell molecular markers revealed remarkable similarity with pathological findings, indicating early and robust neutrophil infiltration, a slight delay in macrophage accumulation, and abundant late populations of T lymphocytes. We also characterized the putative mechanisms regulating a unique, pneumonia-associated biphasic fever pattern. Thus, this study is the first to use a comprehensive and integrative approach to delineate specific molecular mechanisms regulating influenza virus-induced pneumonia in nonhuman primates, an important first step toward better management of human influenza virus disease.

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Since the beginning of the 20th century, humans have experienced four influenza pandemics, including the devastating 1918 'Spanish influenza'. Moreover, H5N1 highly pathogenic avian influenza (HPAI) viruses are currently spreading worldwide, although they are not yet efficiently transmitted among humans. While the threat of a global pandemic involving a highly pathogenic influenza virus strain looms large, our mechanisms to address such a catastrophe remain limited. Here, we show that pre-stimulation of Toll-like receptors (TLRs) 2 and 4 increased resistance against influenza viruses known to induce high pathogenicity in animal models. Our data emphasize the complexity of the host response against different influenza viruses, and suggest that TLR agonists might be utilized to protect against lethality associated with highly pathogenic influenza virus infection in humans.