pinecone/core-2020-05-10-deduplication huggingface dataset:
Characterisation of Gamma Herpesviruses in the Horse by PCR

AbstractA polymerase chain reaction (PCR) based on a combination of oligonucleotide primers selected using the octamer frequency disparity method with primers specific for EHV-5 (described by other authors) recognized all of a series of gamma herpesvirus field isolates. This PCR produced only three fragments: (1) one EHV-2-specific; (2) one EHV-5-specific; and (3) a fragment that occurred alone or in combination with the other two. Cloning and sequencing of four different isolates yielding only the last PCR product showed that this corresponds to a deletion/insertion mutant of EHV-2. The fact that this mutant was also plaque-purified from a culture producing all three PCR fragments demonstrated that the virus producing this fragment was distinct from the other two and that this specific DNA fragment was not an artefact due to PCR amplification. These data show that equine gamma herpesviruses are genetically more heterogeneous than previously assumed. The PCR failed to directly detect gamma herpesviruses from the DNA extracted from the same starting material used for the isolation of gamma herpesvirus by cocultivation with indicator cells. This demonstrates that the most reliable method for detection of equine gamma herpesviruses is the cocultivation with indicator cells
Phylogenetic Analysis of Simian T-lymphotropic Virus Type I (STLV-I) in Common Chimpanzees (Pan troglodytes): Evidence for Interspecies Transmission of the Virus between Chimpanzees and Humans in Central Africa

AbstractSerum and peripheral blood leukocytes from the chimpanzees (Pan troglodytes) of the colony of the Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, NIH, were tested for the presence of STLV-I-specific antibodies and proviral DNA. Antibodies were determined by gelatin particle agglutination and Western blot (WB) assays utilizing HTLV-I antigens. Proviral DNA was detected by four PCR assays targeting three different regions of STLV-I genome: the fragments of theenvandpolgenes andLTR.Twenty of twenty-two DNA samples from WB-positive animals were PCR positive. None of the DNA samples from WB-negative (n= 5) and WB-indeterminate (n= 4) animals was PCR positive. The results of the nested and double nestedenvPCR tests were fully concordant; the seminestedLTRPCR test was much less sensitive. The DNA sequences from theenv(483 bp) and thepol(200 bp) genes andLTR(705 bp) were determined for six, two, and two chimpanzee STLV-I isolates, respectively. Phylogenetic analysis revealed that chimpanzee STLV-I isolates can be attributed to three clades. The first of these clades (SS-PTR1/CSA) included STLV-I isolates from the chimpanzees and West African subspecies of African green monkeys (Cercopithecus a. sabaeus). The other clades (S-PTR2 and S-PTR3) included STLV-I isolates only from chimpanzees. However, both S-PTR2 and S-PTR3 clustered together with Central African HTLV-I comprising the human/simian clade (HS-HSA/PTR). This pattern of phylogenetic clustering suggests that interspecies transmission of STLV-I occurred between chimpanzees and African green monkey subspecies as well between chimpanzees and human populations in Central Africa
Hantavirus Pulmonary Syndrome: CD8+and CD4+Cytotoxic T Lymphocytes to Epitopes on Sin Nombre Virus Nucleocapsid Protein Isolated during Acute Illness

AbstractIn 1993 a number of cases of unexplained adult respiratory syndrome occurred in the southwestern United States. The illness was characterized by a prodrome of fever, myalgia, and other symptoms followed by the rapid onset of a capillary leak syndrome with hemoconcentration, thrombocytopenia, and pulmonary edema. Viral RNA sequences in the lungs identified a new member of the hantavirus genus, Sin Nombre virus (SNV), unique to North America. Pulmonary endothelial cells were heavily infected but were not necrotic. We speculated that this capillary leak syndrome was initiated by immune responses to the SNV-infected pulmonary endothelial cells. We isolated a CD8+cytotoxic T lymphocyte (CTL) clone directly from the blood of a patient with the acute hantavirus pulmonary syndrome (HPS) which recognizes a SNV specific epitope on the virus nucleocapsid protein (aa 234–242) that is restricted by HLA C7 and produces IFNγ but not IL-4. We identified a second CD8+CTL epitope located within another site aa 131–139 on the nucleocapsid protein, which is HLA B35 restricted, and a CD4+CTL epitope located on a third site on nucleocapsid protein aa 372–380 using lymphocytes obtained during HPS from another patient that were stimulatedin vitro.Hantavirus specific CD8+and CD4+CTL may contribute to the immunopathology and capillary leak syndrome observed in the HPS
Construction and Characterization of Vaccinia Direct Ligation Vectors

AbstractPoxvirus vectors are extensively used as expression vehicles for protein and antigen expression in eukaryotic cells. Customarily, the foreign DNA is introduced into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been used to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We describe the construction and characterization of a new set of direct ligation vectors designed to be universally applicable for the generation of chimeric vaccinia genomes. These vectors contain the pair of unique restriction sitesNotI andApaI to eliminate religation of poxvirus arms and fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters. The insertion cassette has been placed at the beginning of the thymidine kinase gene in vaccinia to use drug selection in the isolation of recombinants. These viruses provide a set of universally applicable direct ligation poxvirus cloning vectors, extending the utility of poxvirus vectors for construction and expression of complex libraries
Direct Effect of Type 1 Human Immunodeficiency Virus (HIV-1) on Intestinal Epithelial Cell Differentiation: Relationship to HIV-1 Enteropathy

AbstractHuman immunodeficiency virus (HIV)-infected patients display severe impairments of gastrointestinal functions, including diarrhea and malabsorption, even in the absence of opportunistic infections. Since HIV-1 proteins and nucleic acids have been detected in several cell types of the intestinal mucosa, it has been postulated that HIV-1 itself could alter enterocytic functions. In the present study, we analyzed the effect of HIV-1 on the differentiation process of the epithelial intestinal cell clone HT-29-D4, which mimics the maturation of enterocytes along the crypt–villus axis of the small intestine. We found that HIV-1 infection impairs cellular differentiation (i) by affecting the barrier function of the epithelium, as evidenced by a decrease in the transepithelial electrical resistance, and (ii) by inhibiting the activity of one major glucose absorption function, i.e., sodium/glucose cotransport. At the morphological level, HIV-1 infection of HT-29-D4 cells was associated with the formation of lumina, which are representative of a defect in cellular organization. These morphofunctional perturbations induced by HIV-1 could be mimicked by nocodazole, a microtubule-disrupting agent. Correspondingly, HIV-1 exposure of HT-29-D4 cells evoked a massive disruption of microtubules, as revealed by α-tubulin indirect immunofluorescence staining. A similar effect was observed after incubation of the cells with either recombinant gp120 or a monoclonal antibody against galactosylceramide (GalCer), the intestinal receptor for HIV-1 gp120, suggesting that the effect of HIV-1 was mediated by the binding of gp120 to GalCer. Based on these data, we propose that HIV-1 may selectively alter enterocytic functions through a direct effect on the intracellular architecture of the cells. In contrast with previous theories for HIV-1 enteropathy, our data support the concept that HIV-1 may perturb intestinal functions without necessarily infecting intestinal epithelial cells
Differential Replication of Ovine Lentivirus in Endothelial Cells Cultured from Different Tissues

AbstractBlood–brain barrier dysfunction has been postulated to be important in the pathogenesis of HIV dementia. This study used anin vitromodel of the blood–brain barrier to determine the effects of ovine lentivirus (OvLV) infection on endothelial cells. The replication of two American OvLV isolates and two Icelandic OvLV isolates in pure cultures of endothelial cells isolated from brain was compared to replication in endothelial cells from adipose, lung, and aorta. Inoculation with the two American isolates resulted in 100 times greater reverse transcriptase (RT) activity in supernatant of the microvascular endothelial cells (brain, lung, and adipose) than in the macrovascular endothelial cells (aorta). Conversely, inoculation with the two Icelandic isolates resulted in 100 times higher RT activity in aortic, lung, and adipose endothelial cells than in the brain endothelial cells. Transmission electron microscopy of the brain capillary endothelial cells infected with the American isolates revealed polarized viral budding from the lateral cell membrane and a loss of tight junctions. Replication of OvLV in brain capillary endothelial cells could play a role in the pathogenesis of lentiviral encephalitis by altering blood–brain barrier integrity
Integrin αvβ6 Enhances Coxsackievirus B1 Lytic Infection of Human Colon Cancer Cells

AbstractViral entry into host cells depends upon specific interactions between virus attachment proteins and cell surface receptors that enable virus binding and internalization of virus and/or the virus–receptor complex. We have recently reported that the ubiquitous cell surface molecule, decay-accelerating factor (DAF), is a major cell attachment receptor for Coxsackieviruses B1, B3, and B5. However, DAF permits only virus binding and not virus internalization, invoking the presence of secondary or accessory receptors. Among the known receptors for enteroviruses are members of the cell adhesion molecule family known as integrins. In the present study, we found that expression of the epithelial-restricted integrin, αvβ6, on colonic epithelial cells significantly enhanced Coxsackievirus B1-mediated cell lysis. Importantly, the viral-mediated cell killing required the presence of the 11-amino-acid C-terminal cytoplasmic extension unique to the β6 subunit, providing the first evidence of regulation of viral infectivity by integrin cytoplasmic domains. These results indicate that αvβ6 expression on intestinal epithelial cells critically affects Coxsackievirus B1 infectivity. This may be essential in the conversion of asymptomatic enterovirus infection into clinically apparent disease
Serialin VivoPassage of HIV-1 Infection inMacaca nemestrina

AbstractIn an earlier study we found that pigtailed macaques (Macaca nemestrina) that were experimentally infected with human immunodeficiency virus type 1 (HIV-1) initially became viremic and seroconverted, but HIV-1 replication diminished markedly over time. In an attempt to develop a longer term pathogenic model, blood from HIV-1-infected macaques was serially transfused into three groups of naive macaques. Transfer was successful through two transfusions as shown by repeated virus isolations and confirmed by the development of cell-free plasma viremia and by seroconversion. Three to five weeks after transfusion, plasma levels of HIV-1 RNA from several macaques in the first two groups exceeded those of the initially inoculated macaques. However, animals in the third group had diminished RNA levels, were virus culture negative, and did not seroconvert. Sequence analyses of env-region clones from infected animals revealed only minimal changes over the course of the passages. These results confirm HIV-1 replication inM. nemestrinaduring the acute phase of infection. However, adaptation of HIV-1 to a macaque-pathogenic variant did not occur during serial passage, possibly because the animals were able to restrict HIV-1 replication below a level required for a pathogenic variant to emerge. Whether such containment is a function of the host's immune response or a virus cell incompatibility remains to be determined
Specific Interactions between Human Integrin αvβ3and Chimeric Hepatitis B Virus Core Particles Bearing the Receptor-Binding Epitope of Foot-and-Mouth Disease Virus

AbstractPurified integrin αvβ3was used in solid-phase binding studies with chimeric hepatitis B cores which carry the RGD-containing loop of VP1 protein of the foot-and-mouth disease virus (FMDV). High levels of specific binding between the integrin and the particles were detected by enzyme-linked immunosorbent assays. The binding was Mn2+cation dependent and could be competed with fibronectin, vitronectin, and the peptide GRGDSPK. Particles in which the RGD motif had been mutated to RGE failed to bind, indicating that the chimeric cores bound specifically to the ligand binding site of integrin αvβ3. Electron micrographs showed several individual αvβ3molecules bound to the surface of each chimeric particle. Collectively, these data constitute firm evidence that the RGD-containing loop of FMDV is critical for binding to αvβ3and provide support for identification of αvβ3as a potential cellular receptor for FMDV
Analysis of a Peptide Inhibitor of Paramyxovirus (NDV) Fusion Using Biological Assays, NMR, and Molecular Modeling

AbstractTo investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of a peptide with a 20-amino-acid sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. A peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478–497 of the F protein) was found to inhibit syncytia formation after virus infection and after transfection of Cos cells with the HN (hemagglutinin-neuraminidase) and F protein cDNAs. Using an F protein gene that requires addition of exogenous trypsin for cleavage, it was shown that the peptide exerted its inhibitory effect prior to cleavage. The three-dimensional conformation of the peptide in aqueous solution was determined through the use of NMR and molecular modeling. Results showed that the peptide formed a helix with properties between an α-helix and a 310-helix and that leucine residues aligned along one face of the helix. Side chain salt bridges and hydrogen bonds likely contributed to the stability of the peptide secondary structure. Analysis of the aqueous solution conformation of the peptide suggested mechanisms for specificity of interaction with the intact F protein
Nucleotide Sequence and Genomic Organization of Acyrthosiphon Pisum Virus

AbstractThe nucleotide sequence of the genomic RNA of Acyrthosiphon pisum virus was determined. The APV genome is 10,016 nucleotides in length, excluding the 3′-end poly(A) track, and contains two large open reading frames (ORFs), encoding proteins of 296,340 and 63,279 Da. The ORF1 is preceded by an untranslated leader sequence of 267 nucleotides. The ORF1 product contains sequence motifs characteristic of RNA-dependent RNA polymerases, chymotrypsin-like proteases, and helicases. Interviral sequence comparison revealed significant similarities with viruses belonging to the so-called picornavirus superfamily. The ORF2 is most likely expressed by a −1 translational frameshift and is followed by an untranslated sequence of 222 nucleotides. Internal amino acid sequences of three capsid proteins (66K, 34K, 23/24K) were determined. Comparison of the obtained amino acid sequences with the APV sequence disclosed that the structural proteins are located in the 3′-terminal half of the genome. The 34K protein is encoded by the ORF1, while the 66K protein contains both ORF1- (34K) and ORF2-derived sequences and is probably expressed by a translational frameshift. The 23/24K proteins most likely arise by proteolytic breakdown of the 34K protein. Although the deduced APV genomic organization in some aspects resembles that of the picornaviruses, its overall genomic organization indicates that APV is a distinct species only distantly related to thePicornaviridae
Single Serine Phosphorylation within the Acidic Domain of Chandipura Virus P Protein Regulates the Transcriptionin Vitro

AbstractBacterially expressed unphosphorylated P protein of Chandipura Virus was found to be efficiently phosphorylatedin vitroby casein kinase II (CKII). The phosphorylated form of the P protein supported the transcriptionin vitrobut the unphosphorylated form could not. Kinetic data suggests that CKII incorporates one molecule of phosphate. Western blotting with monoclonal antibody against phosphoserine and phosphoaminoacid analysis confirmed that the phosphate accepting residue was serine. Comparison with P protein of other viruses and tryptic digest of the phosphorylated protein predicted the ser62was the probable site for phosphorylation. This was further confirmed by substituting ser62with alanine by site-directed mutagenesis. CKII was unable to phosphorylate the mutated P protein which in turn could not support the transcriptionin vitro.The phosphorylated P protein eluted from the gel filtration at the position of its dimer in contrast to the unphosphorylated protein which eluted as monomer
Interaction of Endocytic Signals from the HIV-1 Envelope Glycoprotein Complex with Members of the Adaptor Medium Chain Family

AbstractThe envelope glycoprotein (Env) complex of HIV-1 undergoes rapid internalization from the plasma membrane of human cells by virtue of a tyrosine-based endocytic signal (RQGYSPL, residues 704–710) in the cytosolic tail of the protein (J. F. Rowellet al., J. Immunol.155, 473–488, 1995). Here we demonstrate that this tyrosine-based signal interacts with the μ2 (medium) chain of the AP-2 clathrin-associated adaptor, a protein complex involved in endocytosis of cell surface receptors. The same signal is also capable of interacting with two other members of the adaptor medium chain family, μ1 and μ3A, which are components of the AP-1 and AP-3 adaptor complexes, respectively. Interactions with μ1 and μ3A might be responsible for the targeting of the internalized envelope glycoprotein to lysosomes or to the basolateral plasma membrane of polarized epithelial cells. A second potential tyrosine-based signal (LFSYHRL, residues 760–766) also interacts with μ1, μ2, and μ3A, although it is less important for internalizationin vivoprobably due to its position within the cytosolic tail. Overexpression of chimeric proteins having the HIV-1 Env cytosolic tail increases expression of the transferrin receptor on the cell surface, probably due to saturation of the cellular pool of μ2 by the overexpressed proteins. These observations suggest that HIV-1 Env utilizes the protein sorting machinery of the host cells for internalization and sorting at various steps of the endocytic and biosynthetic pathways
Laguna Negra Virus Associated with HPS in Western Paraguay and Bolivia

AbstractA large outbreak of hantavirus pulmonary syndrome (HPS) recently occurred in the Chaco region of Paraguay. Using PCR approaches, partial virus genome sequences were obtained from 5 human sera, and spleens from 5Calomys laucharodents from the outbreak area. Genetic analysis revealed a newly discovered hantavirus, Laguna Negra (LN) virus, to be associated with the HPS outbreak and established a direct genetic link between the virus detected in the HPS cases and in theC. laucharodents, implicating them as the primary rodent reservoir for LN virus in Paraguay. Virus isolates were obtained from twoC. laucha,and represent the first successful isolation of a pathogenic South American hantavirus. Analysis of the prototype LN virus entire S and M and partial L segment nucleotide and deduced amino acid sequences showed that this virus is unique among the Sigmodontinae-borne clade of hantaviruses. Analysis of PCR fragments amplified from a serum sample from a Chilean HPS patient, who had recently traveled extensively in Bolivia (whereC. lauchaare known to occur), revealed an LN virus variant that was approximately 15% different at the nucleotide level and identical at the deduced amino acid level relative to the Paraguayan LN virus. These data suggest that LN virus may cause HPS in several countries in this geographic region
Interferon-γ Protects against Herpes Simplex Virus Type 1-Mediated Neuronal Death

AbstractHost inflammatory mediators, such as interferons, play a protective role in infection, but the mechanism is undefined and may differ between tissue compartments. To determine whether interferon-γ (IFN-γ) elicitation prevents destructive encephalitis in herpes simplex virus type 1 (HSV-1) infection of the central nervous system, IFN-γ-knockout (GKO) mice were challenged intravitreally with HSV-1 strain F, inciting infection of the eyes and the brain. Indeed, the GKO mice showed encephalitis with ataxia, whereas nontransgenic controls remained asymptomatic. Morphology and digoxigenin labeling of DNA fragments revealed increased apoptosis in the brains of GKO mice compared with controls, although viral replication was not influenced at early stages of infection. Greater numbers of apoptotic cells in the brains of GKO mice correlated with neurological symptoms, as well as lower expression of the protective protooncogenebcl-2.Thus, IFN-γ inhibits apoptosis, affording neuronal protection from destructive encephalitis during viral infection of the central nervous system
Efficacy of Replication-Defective Adenovirus-Vectored Vaccines: Protection Following Intramuscular Injection Is Linked to Promoter Efficiency in Muscle Representative Cells

AbstractTo investigate the respective role of transduced cells in the induction of immune response following intramuscular inoculation of adenovirus-based vaccines, we generated several replication-defective adenoviruses expressing the glycoprotein D gene of pseudorabies virus under the control of four different promoters: major late promoter of adenovirus type 2, human cytomegalovirus immediate-early promoter/enhancer (CMV), Rous sarcoma virus-long terminal repeat promoter, and human desmin gene 5′ regulatory region (DES). All the adenovirus constructs were able to fully protect mice, in the contrary of direct DNA inoculation of plasmids harboring the same transcription units. The far most effective adenovirus constructs, on the criterion of protective doses and specific antibody response induction, were those in which the foreign gene was driven by the DES or CMV promoter. Wide variations in promoter strengthin vitrowere evidenced in several cell culture types representative of putative target cells following muscular inoculation (myoblasts, myotubes, fibroblasts, macrophages, and endothelial cells). The level of efficacyin vivo,was not correlated with the level of expressionin vitroin myotubes, but paralleled the level of expression in endothelial cells and in myoblasts. Together with previously published data, these results suggest that, following adenovirus injection, locally produced cytokines may induce myoblasts to act as local antigen presenting cells