Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.
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An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses. (+info)
Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking.
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Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a approximately 40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to alpha-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components. (+info)
Genetic characterization of feline parvovirus sequences from various carnivores.
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Infections with viruses of the feline parvovirus subgroup such as feline panleukopenia virus (FPV), mink enteritis virus (MEV) and canine parvovirus (CPV-2) [together with its new antigenic types (CPV-2a, CPV-2b)] have been reported from several wild carnivore species. To examine the susceptibility of different species to the various parvoviruses and their antigenic types, samples from wild carnivores with acute parvovirus infections were collected. Viral DNA was amplified, and subsequently analysed, from faeces or formalin-fixed small intestines from an orphaned bat-eared fox (Otocyon megalotis), a free-ranging honey badger (Mellivora capensis), six captive cheetahs (Acinonyx jubatus), a captive Siberian tiger (Panthera tigris altaica) and a free-ranging African wild cat (Felis lybica). Parvovirus infection in bat-eared fox and honey badger was demonstrated for the first time. FPV-sequences were detected in tissues of the African wild cat and in faeces of one cheetah and the honey badger, whereas CPV-2b sequences were found in five cheetahs and the bat-eared fox. The Siberian tiger (from a German zoo) was infected with a CPV-type 2a virus. This distribution of feline parvovirus antigenic types in captive large cats suggests an interspecies transmission from domestic dogs. CPV-2 sequences were not detected in any of the specimens and no sequences with features intermediate between FPV and CPV were found in any of the animals examined. (+info)
Comparison of two single-chain antibodies that neutralize canine parvovirus: analysis of an antibody-combining site and mechanisms of neutralization.
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We cloned the heavy- and light-chain variable domains of two monoclonal antibodies that recognize each of the two major neutralizing antigenic sites of the canine parvovirus (CPV) capsid. After expression in Escherichia coli as single-chain variable domains (scFv) with glycine-serine linker sequences, both scFv bound CPV capsids with the same specificity as the intact IgG, but with 10- to 20-fold lower avidity. Both scFvs neutralized CPV infectivity with efficiency similar to that of the IgG. Although both IgGs inhibited hemagglutination by CPV, only one scFv was inhibiting. The binding of one of the antibodies has previously been analyzed by cryoelectron microscopic reconstruction and the epitope-binding residues predicted. Mutagenesis of predicted contact residues in three heavy-chain complementarity-determining regions (CDR) showed that mutants of CDR1 or CDR3 reduced the binding of the scFv by about 10-fold compared with the wild-type scFv, while no effect was seen for one mutant of CDR2. The levels of neutralization of CPV and of hemagglutination inhibition by the scFv mutants were proportional to their reduction in binding affinity compared with the wild type. Neither scFv blocked virus binding to host cells, but they both caused aggregation of the capsids and appeared to affect the process of infection after virus uptake into the cells. (+info)
Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport.
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To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection. (+info)
Parvovirus infection of keratinocytes as a cause of canine erythema multiforme.
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Erythema multiforme major was diagnosed in a dog with necrotizing parvoviral enteritis. Skin lesions consisted of ulceration of the footpads, pressure points, mouth, and vaginal mucosa; vesicles in the oral cavity; and erythematous patches on the abdomen and perivulvar skin. Microscopic examination of mucosal and haired skin specimens revealed lymphocyte-associated keratinocyte apoptosis at various levels of the epidermis. Basophilic cytoplasmic inclusions were seen in basal and suprabasal keratinocytes. Immunohistochemical staining, performed with canine parvovirus-2-specific monoclonal antibodies, confirmed the parvovirus nature of the inclusions in the nucleus and cytoplasm of oral and skin epithelial cells. This is the first case of canine erythema multiforme reported to be caused by a viral infection of keratinocytes. This case study indicates that the search for epitheliotropic viruses should be attempted in cases of erythema multiforme in which a drug cause cannot be identified. (+info)
Predominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats.
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Serological, sequence, and in vitro host range analyses of feline parvovirus (FPV) isolates in Vietnam and Taiwan revealed that more than 80% of the isolates were of the canine parvovirus (CPV) type, rather than feline panleukopenia virus (FPLV). Although parvovirus isolates from three Vietnamese leopard cats were genetically related to CPV type 2a or 2b, they had a natural mutation of VP2 residue 300 Gly to an Asp, resulting in remarkable changes in their antigenic properties. These results indicated the possibility that CPV-2a/2b-type viruses can spread in cats more efficiently than conventional FPLV under natural conditions and that CPV-2a/2b viruses are further evolving in cats. (+info)
Canine parvovirus capsid assembly and differences in mammalian and insect cells.
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We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells. (+info)