Foot-and-mouth disease virus (FMDV) can use a number of different integrins (␣v␤1, ␣v␤3, ␣v␤6, and ␣v␤8) as receptors to initiate infection. Infection mediated by ␣v␤6 is known to occur by clathrinmediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express ␣v␤8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by ␣v␤8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (ϳ35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE. Foot-and-mouth disease virus (FMDV) is a member of theAphthovirus genus of the family Picornaviridae and the etiological agent responsible for FMD, an economically important and severe vesicular condition of cloven-hoofed animals, including cattle, pigs, sheep, and goats (2). The mature virus particle consists of a positive-sense single-stranded RNA genome (vRNA) enclosed within a nonenveloped icosahedral capsid formed from 60 copies each of four virus-encoded proteins, VP1 to VP4 (1).The initial stage of FMDV infection is virus binding to cell surface integrins via a highly conserved RGD motif located on the GH loop of VP1. A number of different species of RGDbinding integrins (␣v␤1, ␣v␤3, ␣v␤6, and ␣v␤8) have been reported to serve as receptors for FMDV (5,(23)(24)(25)(26). Using pharmacological and dominant-negative (DN) inhibitors of specific endocytic pathways in combination with immunofluorescence confocal microscopy, the cell entry pathway used by FMDV has been determined for ␣v␤6-expressing cells (6, 36). These studies established that infection occurs by clathrinmediated endocytosis and is dependent on the acidic pH within endosomes, which serves as the trigger for capsid disassembly and translocation of the vRNA across the endosomal membrane into the cytosol. Internalized virus was detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in l...

For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264.7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH. RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry. However, MNV-1 infection was inhibited by methyl-b-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264.7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA. Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection. These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake. INTRODUCTIONThe family Caliciviridae is divided into four genera: Vesivirus, Lagovirus, Sapovirus and Norovirus. The human noroviruses are the most common cause of acute viral gastroenteritis, especially in industrialized countries (Lopman et al., 2003); there is currently no treatment or vaccine for these viruses. In addition, there is still no routine tissue culture system for the propagation of human noroviruses, but the recent discovery of murine norovirus-1 (MNV-1) has provided an efficient model system for the study of norovirus replication, as this virus replicates efficiently in murine macrophages and dendritic cells (DCs) (Karst et al., 2003;Wobus et al., 2004). MNV-1 is highly prevalent in laboratory mice and has been shown to be lethal to mice with impaired innate immunity (Hsu et al., 2006;Karst et al., 2003;Muller et al., 2007).Noroviruses are non-enveloped and contain a singlestranded RNA genome of about 7.3 kb, which is linked to the viral VPg protein at the 59 end and is polyadenylated at the 39 end (Karst et al., 2003;Wobus et al., 2004). The genome is organized into four open reading frames (ORF-1-4). ORF-1 encodes the non-structural proteins, whereas ORF-2 and ORF-3 encode structural proteins (Sosnovtsev et al., 2006). ORF-4 was recently identified within the MNV-1 genome and encodes a single protein of unknown function (Thackray et al., 2007).For many viruses, infection requires virus uptake by endocytosis. A number of different endo...

SUMMARYCaveolin is a generic term for a family of proteins that include caveolin-1, -2 and -3. Although the distribution of these proteins varies between cells, caveolin-1 and -2 are commonly found coating membrane invaginations known as caveolae. Studies on human and murine cells suggest that caveolin/caveolae can be found in neutrophils, macrophages and mast cells, in which they are involved in the uptake of pathogens, but not in lymphocyte cell lines. Expression of caveolin-1, -2 and -3 in bovine immune cells was investigated using confocal microscopy and Western blotting. Staining for caveolin-1 was evident in all peripheral blood mononuclear cells (PBMC) and in CD4 + , CD8 + and CD21 + lymphocytes, monocytes, macrophages and monocyte-derived dendritic cells (DC). In addition, the caveolin-1 antibody detected a protein with a molecular weight of approximately 22 000 in all PBMC, macrophages and DC, as well as in bovine aortic endothelial (BAE)-1 cells and human endothelial cells by Western blotting. In macrophages and DC, caveolin co-localized with the endoplasmic reticulum±Golgi intermediate compartment (ERGIC) and to a lesser extent with Golgi, but not with endoplasmic reticulum. Staining was not seen on the plasma membrane in any bovine immune cells, suggesting the absence of caveolae, while in BAE-1 cells staining was predominantly on the cell membrane. Caveolin-2 could not be detected in any bovine cells by confocal microscopy or Western blotting, while caveolin-3 was detected in all bovine cells by Western blotting, but not by confocal microscopy. These data provide evidence for the presence of caveolin in bovine lymphocytes and antigen-presenting cells.

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Protection against tuberculosis (TB) is thought to be mediated by Th1‐type responses. Development of antibody responses during TB is associated with disease progression. Development of antibody responses requires the interaction between B‐and T‐cells. To date, the role of B‐cells in the immune response to mycobacteria is not clear. Understanding the role of B‐cells in the immune response to mycobacteria and in particular, their possible role in the development of pathogenic responses, may help develop vaccines that avoid the induction of pathogenic responses and better diagnostics. In this work, we have used the bovine model to study the ability of B‐cells to stimulate mycobacteria‐specific T‐cell responses. We will present evidence that B‐cells are capable of presenting mycobacteria antigens to antigen‐specific CD4+ T‐cells and that the nature of the T‐cell response stimulated by B‐cells differs markedly from T‐cell responses stimulated by macrophages. The implications of the stimulation of CD4+ T‐cell responses by B‐cells, compared to T‐cell responses induced by macrophages will be discussed.

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