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Marshall E. Bloom - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of the small nonstructural proteins of parvovirus Aleutian Mink Disease virus (AMDV) during infection
Virology, 2014Co-Authors: Qinfeng Huang, Marshall E. Bloom, Yong Luo, Fang Cheng, Sonja M. Best, Jianming QiuAbstract:Aleutian Mink Disease virus (AMDV) is the only member in genus Amdovirus of the family Parvoviridae. During AMDV infection, six species of viral transcripts are generated from one precursor mRNA through alternative splicing and alternative polyadenylation. In addition to the large non-structural protein NS1, two small non-structural proteins, NS2 and NS3, are putatively encoded (Qiu J, et al., 2006. J. Virol. 80 654-662). However, these two proteins have not been experimentally demonstrated during virus infection, and nothing is known about their function. Here, we studied the nonstructural protein expression profile of AMDV, and for the first time, confirmed expression of NS2 and NS3 during infection, and identified their intracellular localization. More importantly, we provided evidence that both NS2 and NS3 are necessary for AMDV replication.
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Caspase Cleavage of the Nonstructural Protein NS1 Mediates Replication of Aleutian Mink Disease Parvovirus
Journal of virology, 2003Co-Authors: Sonja M. Best, James B. Wolfinbarger, Janie F. Shelton, Justine M. Pompey, Marshall E. BloomAbstract:Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian Mink Disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD↓S) and NS1:285 (DQTD↓S). Replication of ADV containing either of these mutations was reduced 103- to 104-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.
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Caspase Activation Is Required for Permissive Replication of Aleutian Mink Disease Parvovirus in Vitro
Virology, 2002Co-Authors: Sonja M. Best, James B. Wolfinbarger, Marshall E. BloomAbstract:Abstract Aleutian Mink Disease parvovirus (ADV) is distinct among the parvoviruses as infection in vivo is persistent, restricted, and noncytopathic. In contrast, infections with other more prototypic parvoviruses, like Mink enteritis virus (MEV), are acute, cytopathic, and characterized by permissive replication in vivo. Although apoptosis results in the death of cells acutely infected by parvoviruses, the role of apoptosis in ADV infections is unknown. Permissive infection of ADV resulted in apoptosis of Crandell feline kidney (CrFK) cells as indicated by TUNEL staining, Annexin-V staining, and characteristic changes in cell morphology. Pretreatment of infected cells with caspase 3 or broad-spectrum caspase inhibitors prevented apoptosis. In addition, treatment of infected cells with these inhibitors caused a 2 log 10 reduction in the yield of infectious virus compared to untreated cultures. This block in replication preceded substantial viral DNA amplification and gene expression. However, inhibitors of caspases 1, 6, and 8 did not have this effect. MEV also induced caspase-dependent apoptosis following infection of CrFK cells, although production of infectious progeny was not affected by inhibition of apoptosis. Thus, permissive replication of ADV in vitro depended upon activation of specific caspases. If ADV infection of cells in vivo fails to initiate caspase activation, the requirement of caspase activity for replication may not be met, thus providing a possible mechanism for persistent, restricted infection.
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Aleutian Mink Disease parvovirus in wild riparian carnivores in Spain.
Journal of wildlife diseases, 2001Co-Authors: Sisco Mañas, James B. Wolfinbarger, Juan Carlos Ceña, Jordi Ruiz-olmo, Santiago Palazón, Mariano Domingo, Marshall E. BloomAbstract:Serious declines in populations of native European Mink (Mustela lutreola) have occurred in Europe. One responsible factor may be infectious Diseases introduced by exotic American Mink (Mustela vison). In order to investigate a possible role for Aleutian Mink Disease parvovirus (ADV), we surveyed native riparian carnivores and feral American Mink. When serum samples from 12 free-ranging European and 16 feral American Mink were tested, antibodies to ADV were detected from three of nine European Mink. ADV DNA was detected by polymerase chain reaction in whole cell DNA from four of seven carcasses; two American Mink, one European Mink and a Eurasian otter (Lutra lutra). Lesions typical of Aleutian Disease were present in one of the American Mink. A portion of the ADV VP2 capsid gene was sequenced and the results suggested that two sequence types of ADV were circulating in Spain, and that the Spanish ADVs differed from other described isolates from North America and Europe. Future conservation and restoration efforts should include measures to avoid introduction or spread of ADV infection to native animals.
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Replication of Aleutian Mink Disease parvovirus in Mink lymph node histocultures.
Microbiology, 2000Co-Authors: Klaus T. Jensen, Bent Aasted, James B. Wolfinbarger, Marshall E. BloomAbstract:Aleutian Mink Disease parvovirus (ADV), causes an immune disorder with a persistent infection of lymphoid organs in adult Mink. We studied replication of ADV in gel-supported histocultures prepared from adult Mink mesenteric lymph node (MLN). Evidence of virus replication in the histocultures was first observed by indirect immunofluorescence 72 h after incubation with virus. Cells resembling lymphocytes and macrophages contained both ADV capsid (VP2) and nonstructural (NS1 and NS2) proteins, and were present in a distribution suggestive of infected cells within germinal centres. ADV replicative form and encapsidated virion DNA were also detected in infected histocultures at time-points after 72 h. In addition, we were able to passage ADV-Utah to a new round of histocultures. These results suggested that the infected cells were actual target cells for ADV replication and that productive ADV-Utah replication, complete with the generation of virus, was occurring in the histocultures. The Mink MLN histocultures provide a system to study the replication and molecular pathogenesis of ADV in target tissues.
James B. Wolfinbarger - One of the best experts on this subject based on the ideXlab platform.
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Caspase Cleavage of the Nonstructural Protein NS1 Mediates Replication of Aleutian Mink Disease Parvovirus
Journal of virology, 2003Co-Authors: Sonja M. Best, James B. Wolfinbarger, Janie F. Shelton, Justine M. Pompey, Marshall E. BloomAbstract:Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian Mink Disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD↓S) and NS1:285 (DQTD↓S). Replication of ADV containing either of these mutations was reduced 103- to 104-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.
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Caspase Activation Is Required for Permissive Replication of Aleutian Mink Disease Parvovirus in Vitro
Virology, 2002Co-Authors: Sonja M. Best, James B. Wolfinbarger, Marshall E. BloomAbstract:Abstract Aleutian Mink Disease parvovirus (ADV) is distinct among the parvoviruses as infection in vivo is persistent, restricted, and noncytopathic. In contrast, infections with other more prototypic parvoviruses, like Mink enteritis virus (MEV), are acute, cytopathic, and characterized by permissive replication in vivo. Although apoptosis results in the death of cells acutely infected by parvoviruses, the role of apoptosis in ADV infections is unknown. Permissive infection of ADV resulted in apoptosis of Crandell feline kidney (CrFK) cells as indicated by TUNEL staining, Annexin-V staining, and characteristic changes in cell morphology. Pretreatment of infected cells with caspase 3 or broad-spectrum caspase inhibitors prevented apoptosis. In addition, treatment of infected cells with these inhibitors caused a 2 log 10 reduction in the yield of infectious virus compared to untreated cultures. This block in replication preceded substantial viral DNA amplification and gene expression. However, inhibitors of caspases 1, 6, and 8 did not have this effect. MEV also induced caspase-dependent apoptosis following infection of CrFK cells, although production of infectious progeny was not affected by inhibition of apoptosis. Thus, permissive replication of ADV in vitro depended upon activation of specific caspases. If ADV infection of cells in vivo fails to initiate caspase activation, the requirement of caspase activity for replication may not be met, thus providing a possible mechanism for persistent, restricted infection.
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Aleutian Mink Disease parvovirus in wild riparian carnivores in Spain.
Journal of wildlife diseases, 2001Co-Authors: Sisco Mañas, James B. Wolfinbarger, Juan Carlos Ceña, Jordi Ruiz-olmo, Santiago Palazón, Mariano Domingo, Marshall E. BloomAbstract:Serious declines in populations of native European Mink (Mustela lutreola) have occurred in Europe. One responsible factor may be infectious Diseases introduced by exotic American Mink (Mustela vison). In order to investigate a possible role for Aleutian Mink Disease parvovirus (ADV), we surveyed native riparian carnivores and feral American Mink. When serum samples from 12 free-ranging European and 16 feral American Mink were tested, antibodies to ADV were detected from three of nine European Mink. ADV DNA was detected by polymerase chain reaction in whole cell DNA from four of seven carcasses; two American Mink, one European Mink and a Eurasian otter (Lutra lutra). Lesions typical of Aleutian Disease were present in one of the American Mink. A portion of the ADV VP2 capsid gene was sequenced and the results suggested that two sequence types of ADV were circulating in Spain, and that the Spanish ADVs differed from other described isolates from North America and Europe. Future conservation and restoration efforts should include measures to avoid introduction or spread of ADV infection to native animals.
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Replication of Aleutian Mink Disease parvovirus in Mink lymph node histocultures.
Microbiology, 2000Co-Authors: Klaus T. Jensen, Bent Aasted, James B. Wolfinbarger, Marshall E. BloomAbstract:Aleutian Mink Disease parvovirus (ADV), causes an immune disorder with a persistent infection of lymphoid organs in adult Mink. We studied replication of ADV in gel-supported histocultures prepared from adult Mink mesenteric lymph node (MLN). Evidence of virus replication in the histocultures was first observed by indirect immunofluorescence 72 h after incubation with virus. Cells resembling lymphocytes and macrophages contained both ADV capsid (VP2) and nonstructural (NS1 and NS2) proteins, and were present in a distribution suggestive of infected cells within germinal centres. ADV replicative form and encapsidated virion DNA were also detected in infected histocultures at time-points after 72 h. In addition, we were able to passage ADV-Utah to a new round of histocultures. These results suggested that the infected cells were actual target cells for ADV replication and that productive ADV-Utah replication, complete with the generation of virus, was occurring in the histocultures. The Mink MLN histocultures provide a system to study the replication and molecular pathogenesis of ADV in target tissues.
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Three-dimensional structure of Aleutian Mink Disease parvovirus: implications for Disease pathogenicity.
Journal of virology, 1999Co-Authors: Robert Mckenna, Bent Aasted, Marshall E. Bloom, Paul R. Chipman, Timothy F. Booth, Timothy S. Baker, Norman H. Olson, Jesper Christensen, James M. Fox, James B. WolfinbargerAbstract:The three-dimensional structure of expressed VP2 capsids of Aleutian Mink Disease parvovirus strain G (ADVG-VP2) has been determined to 22 Å resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 Å and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related β-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.
Bent Aasted - One of the best experts on this subject based on the ideXlab platform.
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DISPATCHES Aleutian Mink Disease Virus and Humans
2013Co-Authors: Jørgen R. Jepsen, Michael Roost Clausen, Elisabeth Gottschalck, Bent AastedAbstract:Reports of a possible relationship between Aleutian Mink Disease parvovirus (AMDV) and human infection are rare. However, 2 Mink farmers with vascular Disease and microangiopathy similar to that in Mink with Aleutian Disease were found to have AMDV-specifi c antibodies and AMDV DNA. These fi ndings raise the suspicion that AMDV may play a role in human Disease. Autonomous parvoviruses, such as Aleutian Mink Disease virus (AMDV), cause a broad spectrum of Diseases in animals and man. Acute Disease manifests itself as a lytic infection of rapidly dividing cells; chronic Disease reflects a restricted or abortive infection of specific cell types (1). Aleutian Disease (AD) is known to produce clinical signs in Mink and ferrets only (2,3), although other mammals have reportedly been antibody positive
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Aleutian Mink Disease virus and humans
Emerging infectious diseases, 2009Co-Authors: Jørgen R. Jepsen, Michael Roost Clausen, Elisabeth Gottschalck, Francesco D'amore, Ulrik Baandrup, Bent AastedAbstract:Reports of a possible relationship between Aleutian Mink Disease parvovirus (AMDV) and human infection are rare. However, 2 Mink farmers with vascular Disease and microangiopathy similar to that in Mink with Aleutian Disease were found to have AMDV-specific antibodies and AMDV DNA. These findings raise the suspicion that AMDV may play a role in human Disease.
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Replication of Aleutian Mink Disease parvovirus in Mink lymph node histocultures.
Microbiology, 2000Co-Authors: Klaus T. Jensen, Bent Aasted, James B. Wolfinbarger, Marshall E. BloomAbstract:Aleutian Mink Disease parvovirus (ADV), causes an immune disorder with a persistent infection of lymphoid organs in adult Mink. We studied replication of ADV in gel-supported histocultures prepared from adult Mink mesenteric lymph node (MLN). Evidence of virus replication in the histocultures was first observed by indirect immunofluorescence 72 h after incubation with virus. Cells resembling lymphocytes and macrophages contained both ADV capsid (VP2) and nonstructural (NS1 and NS2) proteins, and were present in a distribution suggestive of infected cells within germinal centres. ADV replicative form and encapsidated virion DNA were also detected in infected histocultures at time-points after 72 h. In addition, we were able to passage ADV-Utah to a new round of histocultures. These results suggested that the infected cells were actual target cells for ADV replication and that productive ADV-Utah replication, complete with the generation of virus, was occurring in the histocultures. The Mink MLN histocultures provide a system to study the replication and molecular pathogenesis of ADV in target tissues.
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Three-dimensional structure of Aleutian Mink Disease parvovirus: implications for Disease pathogenicity.
Journal of virology, 1999Co-Authors: Robert Mckenna, Bent Aasted, Marshall E. Bloom, Paul R. Chipman, Timothy F. Booth, Timothy S. Baker, Norman H. Olson, Jesper Christensen, James M. Fox, James B. WolfinbargerAbstract:The three-dimensional structure of expressed VP2 capsids of Aleutian Mink Disease parvovirus strain G (ADVG-VP2) has been determined to 22 Å resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 Å and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related β-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.
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Epitope mapping of Aleutian Mink Disease parvovirus virion protein VP1 and 2.
Scandinavian journal of immunology, 1999Co-Authors: F Costello, N Steenfos, K T Jensen, J Christensen, E Gottschalck, A Holm, Bent AastedAbstract:Six overlapping fragments of the Aleutian Mink Disease parvoVirus (AMDV) virion protein VP1 and 2 (VP1/2) gene were inserted into the expression vector pMAL-c2. Four of the clones carried large overlapping fragments covering the entire VP1/2 gene. The remaining two clones covered specifically chosen regions within the VP1/2 gene. Using a Western blotting detection system, sera from AMDV-infected Mink were tested against the recombinant polypeptides. These studies showed reactions primarily directed against the two AMDV polypeptides ranging from amino acids 297 to 518. Weaker reactions against other regions of the VP1/2 were also observed. The small fusion protein designed to cover the presumed AMDV VP1/2 loop 4 was purified by affinity chromatography and used to develop solid-phase immunoassays. Twelve small synthetic peptides were constructed and used as inhibitors. A peptide covering amino acids S428 to T448 was shown to block the reactivity of a pool of positive Mink sera, indicating the presence of one dominant linear epitope.
Hongyan Chen - One of the best experts on this subject based on the ideXlab platform.
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Aptamer-targeting of Aleutian Mink Disease virus (AMDV) can be an effective strategy to inhibit virus replication.
Scientific reports, 2021Co-Authors: Hui Zhang, Wenzhuo Yan, Lili Zhao, Jie Zhou, Hongyan ChenAbstract:Aleutian Mink Disease (AMD), which is caused by Aleutian Mink Disease virus (AMDV), is an important contagious Disease for which no effective vaccine is yet available. AMD causes major economic losses for Mink farmers globally and threatens some carnivores such as skunks, genets, foxes and raccoons. Aptamers have exciting potential for the diagnosis and/or treatment of infectious viral Diseases, including AMD. Using a magnetic beads-based systemic evolution of ligands by exponential enrichment (SELEX) approach, we have developed aptamers with activity against AMDV after 10 rounds of selection. After incubation with the ADVa012 aptamer (4 μM) for 48 h, the concentration of AMDV in the supernatant of infected cells was 47% lower than in the supernatant of untreated cells, whereas a random library of aptamers has no effect. The half-life of ADVa012 was ~ 32 h, which is significantly longer than that of other aptamers. Sequences and three dimensions structural modeling of selected aptamers indicated that they fold into similar stem-loop structures, which may be a preferred structure for binding to the target protein. The ADVa012 aptamer was shown to have an effective and long-lasting inhibitory effect on viral production in vitro.
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Development of an antigen-capture enzyme-linked immunosorbent assay for diagnosis of Aleutian Mink Disease virus.
Archives of virology, 2020Co-Authors: Yuanzhi Wang, Lili Zhao, Hongyan ChenAbstract:Aleutian Mink Disease (AMD), caused by Aleutian Mink Disease virus (AMDV), is a very important infectious Disease of Mink. Currently, elimination of antibody- or antigen-positive animals is the most successful strategy for eradicating AMD, but the claw-cutting method of blood sampling is difficult to perform and painful for the animal. In this study, we aimed to establish an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) method for the efficient detection of AMDV antigens using fecal samples. A purified mouse monoclonal antibody (mAb) was used as the capture antibody, and a rabbit polyclonal antibody (pAb) was used as the detection antibody. The assay was optimized by adjusting a series of parameters. Using a cutoff value of 0.205, the limit of detection of the AC-ELISA for strain AMDV-G antigen was 2 μg/mL, and there was no cross-reaction with other Mink viruses. The intra- and inter-assay standard deviations were below 0.046, and the correlation of variance (CV) values were 1.24-7.12% when testing fecal samples. Compared with conventional PCR results, the specificity and sensitivity were 91.5% and 90.6%, respectively, and the concordance rate between the two methods was 91.1%.
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Identification and characterization of a novel B-cell epitope on Aleutian Mink Disease virus capsid protein VP2 using a monoclonal antibody.
Virus research, 2017Co-Authors: Yuanzhi Wang, Wenzhuo Yan, Yuanyuan Zhang, Lili Zhao, Hongyan ChenAbstract:Abstract Aleutian Mink Disease is caused by a highly contagious parvovirus (Aleutian Mink Disease virus, AMDV). This Disease is one of the most commercially important infectious Disease worldwide and causes considerable economic losses to Mink farmers. The capsid protein VP2 is the major immunogenic antigenic protein of AMDV, and is involved in viral tropism, pathogenicity, and host selection. However, few reports have described the use of VP2-specific monoclonal antibodies (mAbs) in B-cell epitope identification and immunological detection. In this study, we produced a specific mAb, 1G5, against AMDV VP2 protein (amino acids: 200 ∼ 588) and characterized its specificity and relative affinity. Six partially overlapping truncated recombinant proteins and seven synthetized peptides were used to identify the epitopes recognized by 1G5. The results indicate that mAb 1G5 can distinguish AMDV, MEV and CPV2 with high affinity (Ka = 5.37 × 109), and the minimal linear epitope is located in amino acid residues 459EEEGWPAASGTHFED473. Sequence alignments demonstrated that the linear epitope was completely conserved among most Amdoparvoviruses except the bat parvovirus, where three substitutions (463W-463F, 466A-466G and 471F-471Y) were noted. Our results reveal that the identified epitope might be a common B-cell epitope of AMDV antibodies, and the 1G5 mAb can be used to identify the cleavage of the capsid proteins during AMDV infection. This is also the first report of a B-cell epitope on AMDV capsid protein VP2 (VP2: 459–473) using a mAb. These findings have potential applications in the development of new diagnostic tools for AMDV.
Soren Alexandersen - One of the best experts on this subject based on the ideXlab platform.
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s phase dependent cell cycle disturbances caused by Aleutian Mink Disease parvovirus
Journal of Virology, 1997Co-Authors: Martin B. Oleksiewicz, Soren AlexandersenAbstract:We examined replication of the autonomous parvovirus Aleutian Mink Disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.
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Subcellular localization of Aleutian Mink Disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells.
Journal of virology, 1996Co-Authors: Martin B. Oleksiewicz, James B. Wolfinbarger, Soren Alexandersen, Fred Costello, Mark E. Huhtanen, Marshall E. BloomAbstract:Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian Mink Disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP proteins formed hollow intranuclear shells around the inclusions. Later, nuclei had irregular outlines and were virtually free of ADV products. In these cells, inclusions of viral DNA with or without associated NS protein were embedded in cytoplasmic VP protein. These findings implied that ADV replication within an infected cell is regulated spatially as well as temporally.
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Investigation of the pathogenesis of transplacental transmission of Aleutian Mink Disease parvovirus in experimentally infected Mink.
Journal of virology, 1996Co-Authors: Susanne Broll, Soren AlexandersenAbstract:The transplacental transmission of Aleutian Mink Disease parvovirus (ADV) was studied in experimental infection of 1-year-old female non-Aleutian Mink. The ADV-seronegative female Mink were inoculated with ADV prior to mating or after the expected implantation of the embryos during pregnancy. A group of uninfected females served as a control group. Animals from each group were killed prior to or shortly after parturition. The in situ hybridization technique with radiolabeled strand-specific RNA probes was used to determine target cells of virus infection and virus replication. In both infected groups, ADV crossed the endotheliochorial placental barrier, although animals infected before mating already had high antibody titers against ADV at the time of implantation. The percentage of dead and resorbed fetuses was much higher in dams infected before mating. In the placentae of these Mink, virus DNA and viral mRNA were detected in cells in the mesenchymal stroma of the placental labyrinth and hematoma but only occasionally in the cytotrophoblast of the placental hematoma. Placentae of animals infected during pregnancy showed in addition very high levels of virus and also viral replication in a large number of cytotrophoblast cells in the placental hematoma, which exhibited distinct inclusion bodies. In both groups, neither virus nor virus replication could be detected in maternal endothelial cells or fetal syncytiotrophoblast of the placental labyrinth. Fetuses were positive for virus and viral replication at high levels in a wide range of tissues. Possible routes of transplacental transmission of ADV and the role of trophoblast cells as targets for viral replication are discussed. Aleutian Mink Disease parvovirus (ADV) in adult Mink causes a persistent infection usually followed by a chronic, progressive Disease associated with a disorder of the immune system (2, 19, 33, 34, 36, 39), which is characterized by the development of plasmacytosis, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis and arteritis in the chronic stage of the Disease. Viral replication was demonstratedinlymphaticorgans,withapeak10daysafterinfection, but then rapidly decreased, and by 60 days after infection, viral replication was below detectable levels (2). InMinkkitsinfectedasnewborns,anacuteinterstitialpneu
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Purification and characterization of the major nonstructural protein (NS-1) of Aleutian Mink Disease parvovirus.
Journal of virology, 1995Co-Authors: Jesper Christensen, Bent Aasted, Michael Pedersen, Soren AlexandersenAbstract:We have previously described the expression of the major nonstructural protein (NS-1) of Aleutian Mink Disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-1 was expressed in Sf9 insect cells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding. ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to Disease in Mink.
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Expression of Aleutian Mink Disease parvovirus proteins in a baculovirus vector system.
Journal of virology, 1993Co-Authors: Jesper Christensen, Soren Alexandersen, Torben Storgaard, Buchardt Bloch, Bent AastedAbstract:Abstract We have previously published a detailed transcription map of Aleutian Mink Disease parvovirus (ADV) and proposed a model for the translation of the two virion structural proteins (VP1 and VP2) and three nonstructural proteins (NS-1, NS-2, and NS-3) (S. Alexandersen, M. E. Bloom, and S. Perryman, J. Virol. 62:3684-3994, 1988). To verify and further characterize this model, we cloned the predicted open reading frames for NS-1, NS-2, NS-3, VP1-VP2, and VP2 alone into a recombinant baculovirus and expressed them in Sf9 insect cells. Expression of VP1-VP2 or VP2 alone in cDNA and in the genomic form was achieved. The expressed proteins had molecular weights similar to those of the corresponding proteins of wild-type ADV-G, although the ratio of VP1 to VP2 was altered. The recombinant baculovirus-expressed ADV VP1 and VP2 showed nuclear localization in Sf9 cells and were able to form particles indistinguishable, by electron microscopy, from wild-type virus. The large nonstructural protein, NS-1, showed predominantly nuclear localization in Sf9 cells when analyzed by immunofluorescence and had a molecular weight similar to that of wild-type ADV NS-1. Moreover, expression of NS-1 in Sf9 cells caused a change in morphology of the cells and resulted in 10-times-lower titers of recombinant baculovirus during infection, suggesting a cytostatic or cytotoxic action of this protein. The smaller NS-2 gene product seems to be located in the cytoplasm. When analyzed by Western immunoblotting, NS-2 comigrated with an approximately 16-kDa band seen in lysates of ADV-infected feline kidney cells. The putative NS-3 gene product exhibited a diffuse distribution in Sf9 cells and had a molecular weight of approximately 10,000. All of the expressed ADV-encoded proteins were recognized by sera from ADV-infected Mink. Thus, expression of ADV cDNAs allowed assignment of the different mRNAs to the viral proteins observed during ADV infection in cell culture and supported our previously proposed ADV transcriptional and translational scheme. Moreover, the production of structural proteins from a full-length NS-2 mRNA may add to the repertoire of parvovirus gene expression.
