bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism
Authors: Escarmís Cristina , Carrillo Elisa C. , Ferrer Marcela , Arriaza Juan F. García , Lopez Nora , Tami Cecilia , Verdaguer Nuria , Domingo Esteban , Franze-Fernández Maria T. 
Published: December 2019

ABSTRACT

With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C
3
Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C
3
Arg/85 infection. Infection of BHK-Rb cells with C
3
Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C
3
-Rb. The selected C
3
-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9→Ala in VP3 and either Gly-110→Arg or His-108→Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C
3
Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells.
Adeno-Associated Virus as a Vector for Liver-Directed Gene Therapy
Authors: Xiao Weidong , Berta Scott C. , Lu Min Min , Moscioni A. David , Tazelaar John , Wilson James M. 
Published: December 2019

ABSTRACT
Factors relevant to the successful application of adeno-associated virus (AAV) vectors for liver-directed gene therapy were evaluated. Vectors with different promoters driving expression of human α-1-antitrypsin (α-1AT) were injected into the portal circulation of immunodeficient mice. α-1AT expression was stable but dependent on the promoter. Southern analysis of liver DNA revealed approximately 0.1 to 2.0 provirus copies/diploid genome in presumed head-to-tail concatamers. In situ hybridization and immunohistochemical analysis revealed expression in approximately 5% of hepatocytes clustered in the pericentral region. These results support the use of AAV as a vector for diseases treatable by targeting of hepatocytes.
In Vitro Construction of Pseudovirions of Human Papillomavirus Type 16: Incorporation of Plasmid DNA into Reassembled L1/L2 Capsids
Authors: Kawana Kei , Yoshikawa Hiroyuki , Taketani Yuji , Yoshiike Kunito , Kanda Tadahito 
Published: December 2019

ABSTRACT
Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a β-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced β-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.
Independent Isolates of the Emerging Subgroup J Avian Leukosis Virus Derive from a Common Ancestor
Authors: Benson Scott J. , Ruis Brian L. , Garbers Amy L. , Fadly Aly M. , Conklin Kathleen F. 
Published: December 2019

ABSTRACT

A new subgroup of avian leukosis virus (ALV) that includes a unique
env
gene, designated J, was identified recently in England. Sequence analysis of prototype English isolate HPRS-103 revealed several other unique genetic characteristics of this strain and provided information that it arose by recombination between exogenous and endogenous virus sequences. In the past several years, ALV J type viruses (ALV-J) have been isolated from broiler breeder flocks in the United States. We were interested in determining the relationship between the U.S. and English isolates of ALV-J. Based on sequence data from two independently derived U.S. field isolates, we conclude that the U.S. and English isolates of ALV-J derive from a common ancestor and are not the result of independent recombination events.
Efficient, Repeated Adenovirus-Mediated Gene Transfer in Mice Lacking both Tumor Necrosis Factor Alpha and Lymphotoxin α
Authors: Benihoud Karim , Saggio Isabella , Opolon Paule , Salone Barbara , Amiot Franck , Connault Elisabeth , Chianale Colette , Dautry François , Yeh Patrice , Perricaudet Michel 
Published: December 2019

ABSTRACTThe efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, and the related cytokine lymphotoxin α (LTα), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-α/LTα−/−), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-α/LTα−/−and TNF-α/LTα+/−mice versus TNF-α/LTα+/+mice links antibody levels to TNF-α/LTα gene dosage. Due to the absence of neutralizing antibodies, the TNF-α/LTα knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-α in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.
Differential Intracellular Compartmentalization of Herpetic Thymidine Kinases (TKs) in TK Gene-Transfected Tumor Cells: Molecular Characterization of the Nuclear Localization Signal of Herpes Simplex Virus Type 1 TK
Authors: Degrève Bart , Johansson Magnus , De Clercq Erik , Karlsson Anna , Balzarini Jan 
Published: December 2019

ABSTRACT

The thymidine kinases (TKs) of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) were expressed in human osteosarcoma cells as fusion proteins with the green fluorescent protein (GFP), and their intracellular localizations were determined. The three TK-GFP fusion products were localized in different subcellular compartments of the transfected tumor cells. HSV-1 TK-GFP was localized exclusively in the nucleus, HSV-2 TK-GFP was predominantly found in the cytosol, while VZV TK-GFP was localized in both the nucleus and the cytosol. In support of these findings, we identified a nuclear localization signal (NLS) in the N-terminal arginine-rich region of HSV-1 TK that was absent in HSV-2 and VZV TK. The first 34 amino acids proved necessary for the specific nuclear localization of HSV-1 TK and, when added to the VZV TK-GFP gene construct, also sufficed to specifically target VZV TK-GFP to the nucleus. Further analysis of this NLS through site-directed mutagenesis revealed that the basic amino acid-rich nonapeptide
25
R-R-T-A-L-R-P-R-R
33
is of crucial importance in the nuclear targeting of HSV-1 TK. In particular, we revealed that the presence of the arginine residues at positions 25, 26, 30, 32, and 33 is obligatory for efficient NLS functioning, whereas arginine and histidine residues outside of the nonapeptide (i.e., residues R18, R20, and H22) did not change the functional properties of the NLS.
Gag Protein Epitopes Recognized by ELA-A-Restricted Cytotoxic T Lymphocytes from Horses with Long-Term Equine Infectious Anemia Virus Infection
Authors: Zhang Wei , Lonning Scott M. , McGuire Travis C. 
Published: December 2019

ABSTRACTMost equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.
Viral Glycoproteins Accumulate in Newly Formed Annulate Lamellae following Infection of Lymphoid Cells by Human Herpesvirus 6
Authors: Cardinali Giorgia , Gentile Massimo , Cirone Mara , Zompetta Claudia , Frati Luigi , Faggioni Alberto , Torrisi Maria Rosaria 
Published: December 2019

ABSTRACT

Ultrastructural analysis of HSB-2 T-lymphoid cells and human cord blood mononuclear cells infected with human herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (AL), which were absent in uninfected cells. Time course analysis of the appearance of AL following viral infection showed that no AL were visible within the first 72 h postinfection and that their formation correlated with the expression of the late viral glycoprotein gp116. The requirement of active viral replication for AL neoformation was further confirmed by experiments using inactivated virus or performed in presence of the viral DNA polymerase inhibitor phosphonoacetic acid. Both conventional electron microscopic examination and immunogold fracture labeling with anti-endoplasmic reticulum antibodies indicated a close relationship of AL with the endoplasmic reticulum and nuclear membranes. However, when the freeze-fractured cells were immunogold labeled with an anti-gp116 monoclonal antibody, AL membranes were densely labeled, whereas nuclear membranes and endoplasmic reticulum cisternae appeared virtually unlabeled, showing that viral envelope glycoproteins selectively accumulate in AL. In addition, gold labeling with
Helix pomatia
lectin and wheat germ agglutinin indicated that AL cisternae, similar to
cis
-Golgi membranes, contain intermediate, but not terminal, forms of glycoconjugates. Taken together, these results suggest that in this cell-virus system, AL function as a viral glycoprotein storage compartment and as a putative site of O-glycosylation.
Cytoplasmic Domain of Sendai Virus HN Protein Contains a Specific Sequence Required for Its Incorporation into Virions
Authors: Takimoto Toru , Bousse Tatiana , Coronel Elizabeth C. , Scroggs Ruth Ann , Portner Allen 
Published: December 2019

ABSTRACT
In the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. The focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. To this end, we examined the specificity of HN incorporation into progeny virions by transiently transfecting HN cDNA genes into Sendai virus (SV)-infected cells. SV HN expressed from cDNA was efficiently incorporated into progeny Sendai virions, whereas Newcastle disease virus (NDV) HN was not. This observation supports the theory of a selective mechanism for HN incorporation. To identify the region on HN responsible for the selective incorporation, we constructed chimeric SV and NDV HN cDNAs and evaluated the incorporation of expressed proteins into progeny virions. Chimera HN that contained the SV cytoplasmic domain fused to the transmembrane and external domains of the NDV HN was incorporated to SV particles, indicating that amino acids in the cytoplasmic domain are responsible for the observed specificity. Additional experiments using the chimeric HNs showed that 14 N-terminal amino acids are sufficient for the specificity. Further analysis identified five consecutive amino acids (residues 10 to 14) that were required for the specific incorporation of HN into SV. These residues are conserved among all strains of SV as well as those of its counterpart, human parainfluenza virus type 1. These results suggest that this region near the N terminus of HN interacts with another viral protein(s) to lead to the specific incorporation of HN into progeny virions.
BiP (GRP78) and Endoplasmin (GRP94) Are Induced following Rotavirus Infection and Bind Transiently to an Endoplasmic Reticulum-Localized Virion Component
Authors: Xu Aimin , Bellamy A. Richard , Taylor John A. 
Published: December 2019

ABSTRACT
Rotavirus infection induces profound alterations in the morphology and biochemistry of the host cell. Using two-dimensional (2D) gel electrophoresis combined with metabolic labeling, we have identified four proteins that are specifically upregulated in rotavirus-infected cells. Two of these have been identified as BiP (GRP78) and endoplasmin (GRP94), members of a family of glucose-regulated chaperone proteins that reside in the endoplasmic reticulum (ER) lumen, the site of rotavirus morphogenesis. The level of mRNA and the transcriptional activity of the BiP and endoplasmin genes are increased markedly in rotavirus-infected cells, and these genes are also induced when a single rotavirus protein, the nonstructural glycoprotein NSP4, is expressed in MA104 cells. However, NSP4 does not associate with either BiP or endoplasmin, implying that the mechanism of BiP and endoplasmin gene activation by NSP4 may differ from that triggered by viral membrane glycoproteins of other viruses. The interaction of BiP and endoplasmin with rotavirus structural polypeptides suggests that these chaperones are involved in the process of viral maturation in the ER lumen.
Formation and Amplification of a Novel Tombusvirus Defective RNA Which Lacks the 5′ Nontranslated Region of the Viral Genome
Authors: Wu Baodong , White K. Andrew 
Published: December 2019

ABSTRACT

Defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) are small, subgenomic, helper-dependent replicons that are believed to be generated primarily by aberrant events during replication of the plus-sense RNA genome. Prototypical TBSV DI RNAs contain four noncontiguous segments (regions I through IV) derived from the 5′ nontranslated region (NTR) (I), an internal section (II), and the 3′-terminal portion (III and IV) of the viral genome. We have studied the formation of these molecules by using engineered precursor DI RNA transcripts and report here the consistent accumulation of a novel defective RNA species, designated RNA B. Northern blot, primer extension, and sequence analyses indicated that, unlike prototypical DI RNAs, RNA B lacks region I. In vitro transcripts corresponding to the region II-III-IV structure of RNA B were amplified when coinoculated with helper, indicating that the 5′ NTR of the genome does not harbor
cis
-acting replication elements essential for viral RNA replication. Region I is, however, important for DI RNA fitness, since molecules lacking it accumulated to significantly lower levels (∼10-fold reduction). Analysis of the minus-strand sequence of region I led to the identification of an RNA undecamer sequence, arranged in tandem, at its very 3′ terminus. Additional variants of the undecamer motif were also identified at internal positions in region I and in the negative strands of regions II, III, and IV. Features of the undecamer motif, the consensus of which is (−)3′-CCCAAAGAGAG, are consistent with a role as a
cis
-acting replication element. It is proposed that the ability of RNA B to be amplified is due, in part, to compensatory effects of a strategically positioned undecamer motif in region II. Possible replicase-mediated mechanisms for the generation of this novel viral RNA are also presented.
Vaccinia Virus Induces Ca
2+
-Independent Cell-Matrix Adhesion during the Motile Phase of Infection
Authors: Sanderson Christopher M. , Smith Geoffrey L. 
Published: December 2019

ABSTRACT

Vaccinia virus (VV) induces two forms of cell motility: cell migration, which is dependent on the expression of early genes, and the formation of cellular projections, which requires the expression of late genes. The need for viral gene expression prior to cell motility suggests that VV proteins may affect how infected cells interact with the extracellular matrix. To address this, we have analyzed changes in cell-matrix adhesion after infection of BS-C-1 cells with VV. Whereas uninfected cells round up and detach from the culture flask in the presence of EGTA, infected cells remain attached to the culture flask with a stellate morphology. Ca
2+
-independent cell-matrix adhesion was evident by 10 h postinfection, after the onset of cell motility but before the formation of virus-induced cellular projections. Progression to Ca
2+
-independent adhesion required the expression of late viral genes but not the formation of intracellular enveloped virus particles or intracellular actin tails. Analyses of specific matrix proteins identified vitronectin and fibronectin as optimal ligands for Ca
2+
-independent adhesion and the formation of cellular projections. Adhesion to fibronectin was mediated via RGD motifs alone and was not inhibited by 500 μg of heparin/ml. Kistrin, a disintegrin which binds preferentially to the αvβ3 (vitronectin/fibronectin) receptor inhibited the formation of cellular projections without disrupting preformed matrix interactions. Finally, we show that Ca
2+
-independent cell-matrix adhesion is a dynamic process which mediates changes in the morphology of VV-infected cells and uninfected cells which exhibit a transformed phenotype.
Protective Role of the Virus-Specific Immune Response for Development of Severe Neurologic Signs in Simian Immunodeficiency Virus-Infected Macaques
Authors: Sopper Sieghart , Sauer Ursula , Hemm Susanne , Demuth Monika , Müller Justus , Stahl-Hennig Christiane , Hunsmann Gerhard , ter Meulen Volker , Dörries Rüdiger 
Published: December 2019

ABSTRACTThe pathogenesis of human immunodeficiency virus-associated motor and cognitive disorders is poorly understood. In this context both a protective and a harmful role of the immune system has been discussed. This question was addressed in the present study by correlating the occurrence of neurologic disease in simian immunodeficiency virus (SIV)-infected macaques with disease progression and the humoral and cellular intrathecal antiviral immune response. Overt neurologic signs consisting of ataxia and apathy were observed at a much higher frequency in rapid progressor animals (6 of 12) than in slow progressors (1 of 7). Whereas slow progressors mounted a strong antiviral antibody (Ab) response as evidenced by enzyme-linked immunosorbent and immunospot assays, neither virus-specific Ab titers nor Ab-secreting cells could be found in the cerebrospinal fluid (CSF) or brain parenchyma of rapid progressors. Similarly, increased infiltration of CD8+T cells and cytotoxic T lymphocytes specific for viral antigens were detected only in the CSF of slow progressors. The finding that neurologic signs develop frequently in SIV-infected macaques in the absence of an antiviral immune response demonstrates that the immune system does not contribute to the development of motor disorders in these animals. Moreover, the lower incidence of neurologic symptoms in slow progressors with a strong intrathecal immune response suggests a protective role of the virus-specific immunity in immunodeficiency virus-induced central nervous system disease.
Host Range and Interference Studies of Three Classes of Pig Endogenous Retrovirus
Authors: Takeuchi Yasuhiro , Patience Clive , Magre Saema , Weiss Robin A. , Banerjee Papia T. , Le Tissier Paul , Stoye Jonathan P. 
Published: December 2019

ABSTRACT

Recent interest in the use of porcine organs, tissues, and cells for xenotransplantation to humans has highlighted the need to characterize the properties of pig endogenous retroviruses (PERVs). Analysis of a variety of pig cells allowed us to isolate and identify three classes of infectious type C endogenous retrovirus (PERV-A, PERV-B, and PERV-C) which have distinct
env
genes but have highly homologous sequences in the rest of the genome. To study the properties of these
env
genes, expression plasmids for the three
env
genes were constructed and used to generate retrovirus vectors bearing corresponding Env proteins. Host range analyses by the vector transduction assay showed that PERV-A and PERV-B Envs have wider host ranges, including several human cell lines, compared with PERV-C Env, which infected only two pig cell lines and one human cell line. All PERVs could infect pig cells, indicating that the PERVs have a potential to replicate in pig transplants in immunosuppressed patients. Receptors for PERV-A and PERV-B were present on cells of some other species, including mink, rat, mouse, and dog, suggesting that such species may provide useful model systems to study infection and pathogenicity of PERV. In contrast, no vector transduction was observed on nonhuman primate cell lines, casting doubt on the utility of nonhuman primates as models for PERV zoonosis. Interference studies showed that the three PERV strains use receptors distinct from each other and from a number of other type C mammalian retroviruses.