The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.The first step in viral entry is the stable attachment of the virion to the surface of a new target cell, a process that can be inefficient for many viruses (16,34,62,73). Cellular proteins that facilitate productive infection by increasing the efficiency of virus binding, but whose presence is not absolutely required for viral entry, are often referred to as attachment factors (4). Two of the most extensively studied attachment factors are the lectins DC-SIGN (CD209) (18, 29) and DC-SIGNR (L-SIGN) (CD209L) (7,67,78). Both are tetrameric type II transmembrane proteins containing calcium-dependent (C-type) carbohydrate recognition domains (CRDs) (55). DC-SIGN is highly expressed in monocyte-derived dendritic cells (MDDCs) in vitro (29) and at lower levels (86) in vivo in subsets of macrophages (45,53,79) and dendritic cells (23,29,40,80,86). DC-SIGNR is expressed on microvascular endothelial cells, especially in the liver sinusoids and lymph nodes (7,67,81). By facilitating virion attachment, DC-SIGN and DC-SIGNR [henceforth referred to collectively as DC-SIGN(R)] can greatly increase the susceptibility of permissive cells to infection by a wide array of enveloped viruses or allow nonpermissive cells to capture and transmit these viruses to target cells in trans (3,17,35,47,52,60,76,84).Viruses that bind to DC-SIGN(R) appear to do so via highmannose, N-linked glycans on their glycoproteins (44,48,51). This fact is readily explained by crystallographic studies demonstrating that mannose-rich oligosaccharides fit into elongated binding sites in the CRDs of DC-SIGN(R) (24). In addition to recognizing viral...

West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.

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West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E).West Nile virus (WNV) is an arthropod-borne virus classified in the Japanese encephalitis antigenic complex of the family Flaviviridae (9, 20, 32). The natural transmission cycle of WNV involves mosquitoes and birds, with humans and other mammals as incidental hosts (8,27). Phylogenetic analysis of WNV strains reveals the presence of two closely related but nonetheless distinct virus groups termed lineage I and lineage II (6). Lineage I strains (which include Kunjin viruses) are distributed worldwide (10,33) and are responsible for all major human outbreaks to date, including the current WNV epidemic in North America (35). In contrast, lineage II WNV strains are restricted to central and southern Africa and do not appear to be as pathogenic as lineage I isolates (6,7,35).WNV contains a single-stranded, plus-sense RNA genome that is translated as a single polyprotein (9). Cleavage of the polyprotein by viral and cellular proteases liberates the viral integral membrane proteins premembrane (prM) and envelope (E), as well as the capsid and seven nonstructural proteins (51). Flavivirus prM and E proteins form heterodimers in the endoplasmic reticulum (ER), where they facilitate virus budding into the ER (2, 40), although one report suggests that the WNV Sarafend strain buds at the plasma membrane (47). As particles travel through the secretory pathway, the bulk of the prM ectodomain is removed by endoproteolysis during transit through the trans-Golgi network (63). Cleavage of prM enables E protein to form head-to-tail homodimers, which form a lattice-like structure covering the surface of the mature, 50 nM diameter virus particle (44). During the process of viral entry, the E protein interacts with an unidentified cell surface receptor(s), followed by uptake into endosomes where the E protein undergoes conformational changes at mildly acid pH, resulting in fusion between the viral and cellular membranes (16,26).All WNV isolates contain a highly conserved N-linked glycosylation site within the ectodomain region of prM that is released during the final stages of particle maturation. In contrast, an N-linked glycosylation site in the E protein at residue 154 is present in many but not all lineage I strains. This site is also present in some lineage II strains, though others contain a 4-amino-acid deletion that ablates this N-linked glycosylation site (1, 6). Interestingly, many of the WNV isolates associated with significant human outbreaks, including the current North American epidemic, contain the N-linked glycosylation site in E (22,35,54). In addition, N-linked glycosylation of the WNV E protein may be associated with altered viral growth in vitro (56) and neuroinvasiveness in a murine model (3,5,61). Nlinked glycosylation of the E protein in WNV and other flaviviruses has been linked to alterations in pH sensitivity (5, 36) and virus yield (65) and likely plays a significant role in the interaction between dengue virus and DC-S...

Indolent lymphoma comprises up to 29% of all canine lymphoma; however, limited information exists regarding the subtypes and biological behaviour. This retrospective study describes the clinical characteristics, histopathological and immunohistochemical features, treatment, outcome and prognostic factors for 75 dogs with indolent lymphoma. WHO histopathological classification and immunohistochemistry (IHC) for CD79a, CD3, Ki67 and P-glycoprotein (P-gp) was performed. The most common histopathological subtype was T-zone, 61.7%, (MST 33.5 months), followed by marginal zone, 25%, (MST 21.2 months), P = 0.542. The addition of IHC to preliminary histopathological classification resulted in a revised diagnosis in 20.4% of cases. The use of systemic treatment did not influence survival, P = 0.065. Dogs treated with chlorambucil and prednisone did not reach a MST, compared with a MST of 21.6 months with CHOP-based chemotherapy, P = 0.057. The overall MST of 4.4 years confirms that this is indeed an indolent disease. However, the effect of systemic treatment must be determined through prospective trials.

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.

We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.

Although many coxsackie B viruses interact with decay accelerating factor (DAF), attachment to DAF by itself is not sufficient to initiate infection. We examined the early events in infection that follow virus interaction with DAF, and with the coxsackievirus and adenovirus receptor (CAR). Interaction with soluble CAR in a cell-free system, or with CAR on the surfaces of transfected cells, induced the formation of A particles; interaction with soluble or cell surface DAF did not. The results suggest that CAR, but not DAF, is capable of initiating the conformational changes in the viral capsid that lead to release of viral nucleic acid.