Summary Avian influenza A viruses rarely infect humans, but if they do and transmit among them, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern due to the appreciable case fatality rate associated with these infections (>25%), potential instances of human-to-human transmission1, and the lack of pre-existing immunity among humans to viruses of this subtype. Here, we therefore characterized two early human A(H7N9) isolates, A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9; hereafter referred to as Anhui/1 and Shanghai/1, respectively). In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011; H7N9; Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/04/2009; H1N1; CA04). Anhui/1, Shanghai/1, and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates (NHPs), Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs upon intranasal inoculation. Most critically, Anhui/1 transmitted via respiratory droplets in one of three pairs of ferrets. Glycan arrays demonstrated that Anhui/1, Shanghai/1, and A/Hangzhou/1/2013 (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was less sensitive than a pandemic 2009 H1N1 virus to neuraminidase inhibitors, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets, and NHPs and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours (‘Color-flu’ viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.

The liver and activation-regulated chemokine (LARC), also termed MIP-3 § and Exodus, is a novel human CC chemokine with a selective chemotactic activity for lymphocytes and den-dritic cells. Here we describe genomic and cDNA clones encoding the murine orthologue of LARC (mLARC). The gene consists of four exons and three introns. The 5'-noncoding region of about 400 bp contains typical TATA and CAAT boxes but no other potential regulatory elements so far described. The cDNA encodes a CC chemokine of 97 amino acid residues with the highest homology to human LARC (64 % amino acid identity). The 3'-noncoding region contains as many as five potential mRNA destabilization signals. mLARC was strongly and transiently induced in the murine monocytoid cell line J774 by lipopolysaccharide (LPS) but not by cytokines such as TNF-§ , IFN-+ , IL-1 g or IL-4. In normal mice, mLARC mRNA was expressed selectively in intestinal tissues such as small intestine and colon. Upon treatment with LPS, mLARC expression was enhanced in intestinal tissues and induced in some lym-phoid tissues such as lymph nodes. Because of alternative splicing, there are two types of transcripts encoding mLARC and its variant mLARCvar with and without an N-terminal ala-nine in the mature protein, respectively. Both types of transcripts appeared to be expressed in various mouse tissues. In situ hybridization revealed that epithelial cells of intestinal tissues , especially those lining lymphoid follicles, expressed mLARC. Localization of LARC mRNA in epithelial cells was also demonstrated in a human appendix. Furthermore, mLARC was efficiently chemotactic for cells such as + ˇ type T cells in intestinal epithelium and naive B cells in Peyer's patches. Thus, in both humans and mice, LARC may be physiologically involved in formation and function of the mucosal lymphoid tissues by attracting lympho-cytes and dendritic cells toward epithelial cells.

Sphingosine 1-phosphate (S1P) is known to play a pivotal role in the regulation of lymphocyte emigration from organized lymphoid tissues such as the peripheral lymph nodes and thymus, but its immunologic role in unorganized and diffused tissues remains to be elucidated. Here we show that the trafficking of peritoneal B cells is principally regulated by S1P. All peritoneal B cells including B1a, B1b, and B2 B cells express comparable levels of the type 1 S1P receptor. Thus, treatment with FTY720, an S1P receptor modulator, caused the rapid disappearance of peritoneal B cells by inhibiting both their emigration from parathymic lymph nodes and their recirculation from the blood into the peritoneal cavity without affecting their progenitor populations. These changes did not affect natural plasma antibody production or phosphorylcholine (PC)-specific antibody production in serum after peritoneal immunization with heatkilled Streptococcal pneumoniae (R36A). However, FTY720 dramatically reduced peritoneal B cell-derived natural intestinal secretory IgA production without affecting the expression of J-chain and polyimmunoglobulin receptors. Additionally, FTY720 impaired the generation of PC-specific fecal IgA responses after oral immunization with R36A. These findings point to a pivotal role for S1P in connect IntroductionSphingosine 1-phosphate (S1P) has been identified as an important molecule in the regulation of lymphocyte egress from the organized lymphoid structures including the thymus and secondary lymphoid organs. 1,2 At present, 5 kinds of S1P receptors have been identified, each sharing S1P as its ligand but associating with a different type of G protein, resulting in a distinct signal transduction. 3 Accumulating evidence has demonstrated that type 1 S1P receptor (S1P1) is preferentially expressed on lymphocytes, and their expression is closely regulated by lymphocyte activation or development, which determines lymphocyte emigration from secondary lymph organs as well as the thymus. 4,5 FTY720, 2-amino-2-[2-(4-octylphenyl)-ethyl] propane-1,3-diol hydrochloride, acts as an agonist for S1P receptors, except the type 2 S1P receptor (S1P2). 6-8 FTY720 blocks S1P-mediated signaling by inducing internalization of receptors. 4,9 Therefore, treatment with FTY720 decreased the number of circulating lymphocytes in both blood and lymph, by inhibiting their emigration from the secondary lymphoid organs and thymus and by modulating integrin-dependent lymphocyte homing into peripheral lymph nodes. [10][11][12] Several lines of evidence have revealed that S1P also regulates B-cell distribution in the spleen, suggesting that FTY720 can impair plasma antibody production, especially against T-dependent (TD) antigen due to the abolishment of germinal center formation. [13][14][15] In addition, a recent study revealed that S1P plays an important role in the determination of plasma cell tropism to bone marrow. 16 Despite the substantial evidence pointing to the role of S1P in the regulation of lymphocyte trafficking at the syst...