Melissa D. Sánchez

West Nile Virus Discriminates between DC-SIGN and DC-SIGNR for Cellular Attachment and Infection

ABSTRACT 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.

DC-SIGN Interactions with Human Immunodeficiency Virus Type 1 and 2 and Simian Immunodeficiency Virus

ABSTRACT Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.

N-Linked Glycosylation of West Nile Virus Envelope Proteins Influences Particle Assembly and Infectivity

ABSTRACT West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.

CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity

ABSTRACT 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.

Interaction with Coxsackievirus and Adenovirus Receptor, but Not with Decay-Accelerating Factor (DAF), Induces A-Particle Formation in a DAF-Binding Coxsackievirus B3 Isolate

ABSTRACT 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.

Clinical, histopathological and immunohistochemical characterization of canine indolent lymphoma.

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.

Tissue-Specific Deletion of the Coxsackievirus and Adenovirus Receptor Protects Mice from Virus-Induced Pancreatitis and Myocarditis

In cultured cells, infection by group B coxsackievirus (CVB) is mediated by the coxsackievirus and adenovirus receptor (CAR), but the importance of this molecule in CVB-induced disease has not been determined. We generated mice with tissue-specific ablation of CAR within each of two major CVB target organs, the pancreas and heart. In the pancreas, deletion of CAR resulted in a significant reduction in both virus titers and virus-induced tissue damage. Similarly, cardiomyocyte-specific CAR deletion resulted in a marked reduction in virus titer, infection-associated cytokine production, and histopathology within the heart. Consistent with the in vivo phenotype, CAR-deficient cardiomyocytes resisted infection in vitro. These results demonstrate a critical function for CAR in the pathogenesis of CVB infection in vivo and in virus tropism for the heart and pancreas.

A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection

Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N-linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain.

Multifocal Oligodendroglioma in Three Dogs

This report describes the clinical, histopathologic, and imaging findings of multifocal oligodendrogliomas from three canine patients. Clinical history varied but included seizure activity and behavior changes. Neurologic examination abnormalities included ataxia, proprioceptive deficits, cranial nerve deficits, and changes in mentation. MRI in one patient revealed multifocal brain lesions; however, the MRI was normal in another one of the patients. Histopathologic evaluation identified multifocal neoplastic infiltrates in all three patients involving the cerebral cortex, brainstem, and spinal cord, with leptomeningeal extension in two of the three patients. All three patients were euthanized due to progression of their neurologic condition and/or complications due to aspiration pneumonia. Oligodendrogliomas should be considered a differential diagnosis for patients with multifocal brain disease.

Herpesvirus dermatitis in two cats without facial lesions

BACKGROUND Cats with feline herpesvirus (FeHV-1)-associated dermatitis typically present with ulcerative lesions on the rostral muzzle and nasal planum. This report describes FeHV-1 dermatitis in the flank region, in the absence of facial lesions. HYPOTHESIS/OBJECTIVES Clinicians should be aware of this unusual manifestation of FeHV-1 dermatitis to prevent potential misdiagnosis. ANIMALS A 12-year-old male castrated Bengal cat and a 3-year-old male castrated Siamese cat with plaques and ulcers in the flank region are described. METHODS Formalin-fixed biopsy samples were obtained from lesional skin. Histopathology and FeHV-1 immunohistochemistry were performed. RESULTS Each sample had epidermal and follicular necrosis with a dense dermal infiltrate of eosinophils. Few to moderate numbers of intranuclear inclusion bodies were present in keratinocytes. The presence of FeHV-1 in the lesions was confirmed with immunohistochemistry. CONCLUSIONS AND CLINICAL IMPORTANCE Feline herpesvirus-associated dermatitis should not be ruled out based on the location of the lesion, because a correct diagnosis is imperative for proper treatment. Future studies to assess the cause of lesions at this unusual site are warranted.