Development of a monoclonal antibody against canine parvovirus NS1 protein and investigation of NS1 dynamics and localization in CPV-infected cells.

Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.

Canine morbillivirus (canine distemper virus) with concomitant canine adenovirus, canine parvovirus-2, and Neospora caninum in puppies: a retrospective immunohistochemical study.

A retrospective immunohistochemical study was designed to investigate the frequency of concomitant traditional infectious disease pathogens in puppies that died suddenly and review the aspects of associated pathogenesis. Fifteen puppies were evaluated; the pathology reports and histopathologic slides of these animals were reviewed to determine the pattern of histopathologic lesions. The intralesional identification of antigens of canine (distemper) morbillivirus (CDV), canine adenovirus-1 and -2 (CAdV-1 and -2), canine parvovirus-2 (CPV-2), Toxoplasma gondii, and Neospora caninum was evaluated by IHC within the histopathologic patterns observed. All puppies contained CDV nucleic acid by molecular testing. The most frequent histopathologic patterns were intestinal crypt necrosis (n = 8), white matter cerebellar demyelination (n = 7), necrohaemorrhagic hepatitis (n = 7), interstitial pneumonia (n = 7), and gallbladder oedema (n = 5). All puppies contained intralesional antigens of CDV in multiple tissues resulting in singular (n = 3), and concomitant dual (n = 3), triple (n = 5) and quadruple (n = 4) infections by CAdV-1, and -2, CPV-2, and N. caninum; T. gondii was not identified. Concomitant infections by CDV was observed with N. caninum (100%; 1/1), CPV-2 (100%; 8/8), CAdV-1 (100%; 8/8), and CAdV-2 (100%; 8/8). Intralesional antigens of CDV and not CAdV-1 were identified in cases of gallbladder oedema. The "blue eye" phenomenon was histologically characterized by corneal oedema and degenerative lesions to the corneal epithelium, without inflammatory reactions.

Viral highway to nucleus exposed by image correlation analyses.

Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin ß revealing previously unknown dynamics of the entry pathway.

In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

The canine Parvovirus 2, non-structural 1 (NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1 (rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS-PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 x His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector.

For the potential use as recombinant vaccine, canine parvovirus (CPV) major capsid protein VP2 was expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) vector. CPV VP2 gene was introduced into polyhedrin-based BmNPV transfer vector pBmKSK3, and recombinant virus BmK1-Parvo was prepared. When anti-CPV.VP2 monoclonal antibody was employed in immunofluorescence staining, an intense signal was observed within BmK1-Parvo-infected Bm5 cells but not within uninfected cells or cells infected with a wild-type BmNPV-K1. In hemagglutination assay, the expression level of VP2 were 3.2 x 10(3) HA units/ml from infected Bm5 cells, 2.1x 10(5) HA units/larvae from infected larval fat body, and 1.6x 10(6) HA units/ml from infected larval hemolymph. These results suggested that BmNPV vector system using B. mori larva as host could be applied to efficient mass-production of recombinant vaccines.

Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking.

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.

Identification of a 40- to 42-kDa attachment polypeptide for canine parvovirus in A72 cells.

The attachment of canine parvovirus (CPV) to different cell lines was quantitated by a fluorescence-activated cell sorter assay. The viral attachment was observed to both permissive A72 and nonpermissive ST cells but not to nonpermissive MDBK cells. The binding of and infectivity for CPV to A72 cells was reduced upon prior treatment of cells with Vibrio cholerae neuraminidase or lectins, specific for sialic acid. Similarly, treatment of cells with any of several proteases reduced virus binding; however, phospholipase treatment had no effect indicating that one or more membrane glycoproteins were involved in virus binding. These proteins were characterized with a virus overlay protein blot assay. Virus bound to a protein with a molecular mass of 40 to 42 kDa in membranes prepared from A72 and ST cells and not from MDBK cells. The binding to this polypeptide was specific since increasing amounts of unlabeled virions competitively inhibited binding of radiolabeled virions in a dose-dependent manner. A polypeptide of similar molecular mass was immunoprecipitated from radiolabeled octyl glucoside (OG) extract of A72 cells using purified virions, virion-specific antiserum, and protein A. The binding to this polypeptide was decreased but not abolished upon prior treatment of the membrane with V. cholerae neuraminidase. CPV preferentially recognized a polypeptide of similar molecular size in the OG extract prepared from the biotinylated basolateral surface of polarized MDCK monolayer. Hence, we propose that the 40- to 42-kDa glycoprotein represents a specific attachment molecule for CPV in A72 cells.