pinecone/core-2020-05-10-deduplication huggingface dataset:
Generation and Characterization of Organ-Tropism Mutants of Japanese Encephalitis Virusin Vivoandin Vitro

AbstractUsing γ-ray irradiation, a pair of virulent (RP-9) and attenuated (RP-2ms) variants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate, NT109. The two variants differed in plaque morphology, virus adsorption, and growth properties in BHK-21 cells: (i) RP-2ms produced smaller plaques than RP-9; (ii) RP-2ms adsorbed less efficiently to host cells but yielded a higher virus titer (burst size); and (iii) RP-2ms virions were mostly accumulated intracellularly, whereas RP-9 was released extracellularly. In addition, in anin vitrobinding assay, the envelope (E) protein of RP-9, but not that of RP-2ms, bound specifically to a cellular protein of 57-kDa derived from BHK-21 cells. When injected into mice intracerebrally, RP-2ms was much less virulent than RP-9, with 50% lethal doses of >107and 0.4 plaque forming units, respectively. Moreover, when inoculated intraperitoneally, their organ tropism differed in that the main target organ for RP-2ms was liver, whereas that for RP-9 was brain. These results suggest that RP-2ms was less neurovirulent and less neuroinvasive from peripheral routes. Molecular analysis of the virus structural proteins detected only two differences between RP-9 and RP-2ms: one in E protein, Glu-138 in RP-9 and Lys-138 in RP-2ms, and the other in prM, Tyr-43 in RP-9 and His-43 in RP-2ms. Since the N-terminal 92 amino acids of prM are cleaved and not present in mature JEV virions, the single-amino-acid change of the E protein at position 138 may account for the difference between the mutants in thein vitrobinding assay. Such mutation in E protein, or perhaps in conjunction with the prM mutation, may be responsible, in part, for the phenotypic differences observedin vitroandin vivobetween the two mutants
The Evolutionary Rate of Nonpathogenic Simian Immunodeficiency Virus (SIVagm) Is in Agreement with a Rapid and Continuous Replicationin Vivo

AbstractAfrican green monkeys (AGMs) are divided into four species (Cercopithecus aethiops, C. pygerythrus, C. sabaeus, C. tantalus), each harboring a species-specific simian immunodeficiency virus (SIVagm). Little is known about the host and/or viral factors that are responsible for the apathogenicity of SIVagm infections in their natural hosts. In order to analyze the specific selective pressures exerted by the host on the virusin vivo,we compared the genetic evolution of SIVagm.tan in its natural host (C. tantalus) and in a foreign host species (Erythrocebus patas), in which we could obtain a reproducible and persistent infection by SIVagm.tan. As in AGMs, patas monkeys do not develop any disease following SIVagm infection. Our longitudinal study inenv(V3-C3-V4-C4-V5) of SIVagm.tan from three AGMs and three patas monkeys revealed a high ratio of synonymous to nonsynonymous mutation frequencies (1.5–6.2). These data indicate that the selective pressures for stability exerted by AGMs and patas monkeys on SIVagm override positive selection for change reported in pathogenic HIV-1 infections. The rapid accumulation of mutations observed in AGMs and patas monkeys (0.4–7.2 × 10−2nucleotide substitutions per site per year) suggests a continuous replication of SIVagm virusesin vivo.We thus propose that nonpathogenic SIVagm infections are the result of a long-term selection of SIVagm variants whose dissemination can be controlled in the host, rather than being explained by a low ability of the virus to replicatein vivo
Peptides Corresponding to the Heptad Repeat Sequence of Human Parainfluenza Virus Fusion Protein Are Potent Inhibitors of Virus Infection

AbstractIt has been suggested that a conserved heptad repeat region in paramyxovirus fusion (F) proteins is essential for viral fusion activity (Bucklandet al.,1992; Sergelet al.,1994; Reitteret al.,1995). We have studied synthetic peptides containing the heptad repeat regions derived from the F proteins of human parainfluenza virus type 2 (PI2) and type 3 (PI3) for their function as potential inhibitors of virus-induced cell fusion as well as their effects on spread of viral infection. Two peptides containing sequences of heptad repeat B, adjacent to the transmembrane domain of the F protein, were synthesized for both PI2 and PI3 F proteins. We observed that the longer peptides [34 amino acids (a.a.) for PI2F or 35 a.a. for PI3F] which extend from heptad repeat B to the transmembrane domain showed complete inhibition of cell fusion induced by the respective virus as well as by the vaccinia-expressed F and HN proteins. The 50% effective concentration to inhibit virus-induced cell fusion was 2.1 μMfor PI2 and 1.2 μMfor PI3. Moreover, the inhibitory effects of each peptide on virus-induced cell fusion were found to be virus type-specific. These peptides were found to also inhibit viral entry and to prevent plaque formation when mixed with the virus inoculum. Furthermore, the peptides caused a reduction in virus yield when assayed 48 hr after low m.o.i. infection and in the size of viral plaques when added to the overlay. Shorter peptides (21 a.a. for PI2F or 24 a.a. for PI3F) which correspond to the partial sequence of heptad repeat B for PI2F and the entire heptad repeat B for PI3F showed partial inhibition of PI2- or PI3-induced cell fusion. These results indicate that peptides containing the heptad repeat B sequence have the potential to inhibit virus-induced cell fusion, virus entry, and spread of virus infection
Determination of Occupancies of the SPH and GT-IIC Transcription Factor Binding Motifs in SV40: Evidence for Two Forms of Transcription Elongation Complex

AbstractOccupancies of the SPH and GT-IIC sequence motifs in the native SV40 late transcription elongation complex were determined by assessing blockage to restriction enzyme cleavage. Cleavages specific to the transcription elongation complex were quantified by radioactive extension labeling and polymerase run-off analysis. The SPH motif was assayed bySphI digestion and found to be unoccupied. In contrast, digestion withPvuII at the GT-IIC site was blocked in 36% of the complexes, indicating that approximately a third of the complexes are occupied by factor. This fractional occupancy indicates that there are at least two forms of SV40 late transcription elongation complexes, one form with the GT-IIC site occupied by a factor and another with the site vacant
Cowpox Virus Genome Encodes a Second Soluble Homologue of Cellular TNF Receptors, Distinct from CrmB, That Binds TNF but Not LTα

AbstractWe show the cowpox genome (Brighton Red strain) contains a single copy gene,crmC,expressed at late times during viral infection, encoding a soluble, secreted protein whose sequence marks it as a new member of the TNF receptor family. The cysteine-rich protein contains 186 amino acids, the N-terminal 21 of which constitute a signal peptide, and two potential N-linked glycosylation sites. The ∼25-kDa recombinant protein binds TNF specifically and completely inhibits TNF-mediated cytolysis. The strongest sequence homologues are the ligand-binding regions of the type II cellular TNF receptor (TNFRII) and CrmB, a distinct pox virus gene also encoding a soluble TNF binding protein. Unlike TNFRII and CrmB, CrmC does not bind lymphotoxin (LTα, TNFβ) and lacks the conserved (but nonhomologous) ∼150-residue C-terminal domain of CrmB proteins. The presumed function of CrmC is viral inhibition of host-elicited TNF
Genetic Diversity in Tobacco Mild Green Mosaic Tobamovirus Infecting the Wild PlantNicotiana glauca

AbstractThe variability and genetic structure of tobacco mild green mosaic tobamovirus (TMGMV) was analyzed in a collection of isolates fromNicotiana glaucaplants from Australia, California, Spain, and the east Mediterranean Basin. Ribonuclease protection was assayed on 53 isolates with six probes representing 85% of the TMGMV genome. Results showed that conserved domains in the TMGMV genome were different for each geographic population. The nucleotide sequence of two regions of the TMGMV genome was determined for 33 isolates. Nucleotide diversity values were smaller than those reported for other RNA genomes. For each population, genetic diversity was not related to the size of the area from which the isolates were collected, and was particularly small for Australia and Spain. Diversity values indicated population differentiation, which was largest between the less diverse Spanish and Australian populations. Nucleotide diversity values for nonsynonymous vs synonymous positions indicate moderate negative selection, except for the Spanish population for which it gave evidence of positive selection. On the whole, our data indicate that the low genetic diversity for this plant virus may be due to different factors such as positive and negative selection or the recent colonization of new areas or host plants. The intensity of these factors appears to be different for the various TMGMV geographical populations
Isolation of Recombinant Viruses between Cauliflower Mosaic Virus and a Viral Gene in Transgenic Plants under Conditions of Moderate Selection Pressure

AbstractWe demonstrate that recombinant viruses formed between a wild-type virus and a viral transgene can be isolated from transgenic plants under conditions of moderate to weak selection pressure. We inoculated cauliflower mosaic virus (CaMV) strain W260 to transgenicNicotiana bigeloviiplants that expressed a copy of CaMV gene VI derived from CaMV strain D4, a gene that determines systemic infection of solanaceous species, includingN. bigelovii.Because W260 infects nontransformedN. bigeloviisystemically, a recombinant virus formed between W260 and the D4 transgene would be expected to have little selective advantage over the wild-type W260 virus. W260 was inoculated to approximately 100 plants each of nontransformed and transgenicN. bigeloviiand it systemically infected nearly all of the plants. An analysis of viral DNA recovered from 23 transgenic plants infected with W260 revealed that 20 infections resulted from the systemic movement of the wild-type W260 virus, while a recombinant between W260 and the D4 transgene was detected in three of the infections. To determine the percentage of recovery of recombinant viruses under strong selection pressure, we inoculated approximately 100 nontransformed and 100 D4 gene VI transgenic plants with CaMV strain CM1841, a virus that is unable to infect nontransformedN. bigelovii.CM1841 infected 36% of the transgenic plants systemically, but none of the nontransformed controls. An analysis of 24 infected plants showed that a recombination event occurred in every plant, demonstrating that under strong selection conditions, the recovery of CaMV recombinants from transgenic plants can be very high
Surface Conformational and Linear Epitopes on HPV-16 and HPV-18 L1 Virus-like Particles as Defined by Monoclonal Antibodies

AbstractA panel of 24 monoclonal antibodies (MAbs) was generated against human papillomavirus (HPV) types 16 and 18 L1 virus-like particles (VLPs). The MAbs were screened for reactivity to a variety of VLPs prepared from HPV-6, -11, -16, -18, -31, -33, -35, and -45, cottontail rabbit papillomavirus, bovine papillomavirus type 1, and a set of 35 overlapping 20-amino-acid peptides spanning the entire HPV-16 L1 gene. Type-specific linear and conformational surface epitopes were detected as well as several cross-reactive linear epitopes that showed various levels of cross-reactivity between different genital HPV and animal papillomavirus L1s. Most of the linear epitopes were mapped using synthetic peptides, and the epitopes were identified as being either surface or buried within the VLP as defined by the pattern of reactivity in ELISA using intact and disrupted VLP antigen. These MAbs may be useful reagents to help define neutralizing epitopes of HPV-16 and -18 when infectivity assays become available, and to define the regions of L1 that are exposed on the surface or buried within the assembled capsid
HPV6 Variants from Malignant Tumors with Sequence Alterations in the Regulatory Region Do Not Reveal Differences in the Activities of the Oncogene Promoters but Do Contain Amino Acid Exchanges in the E6 and E7 Proteins

AbstractHuman papillomavirus type 6 (HPV6) causes benign epithelial proliferations of the anogenital and aerodigestive tract, which usually tend to regress spontaneously. The low incidence of HPV6 in carcinomas and the rare progression of the benign tumors has led to the classification of HPV6 as “low-risk” virus. A series of reports, however, described the isolation of HPV6 variants from malignant tumors characterized by sequence rearrangements in the noncoding regulatory region (NCR). It was speculated that these sequence alterations play a role in tumor progression by enhancing the promoter activity and thereby increasing the expression of the viral oncogenes E6 and E7. To elucidate if HPV6 isolates from malignancies do regularly exhibit sequence alterations in the regulatory region we first determined and compiled the sequences of the NCRs of a number of isolates from benign and malignant lesions. This analysis revealed in general a high degree of sequence conservation between the individual isolates. Most of the isolates, however, differed, independently of origin, by a major and one or two minor insertions from the prototype HPV6b sequence. When tested in a functional assay these altered NCR sequences did not result in significantly different activities of the promoters responsible for the expression of the E6 and E7 genes. Further analysis of the E6 and E7 coding region revealed a surprisingly high sequence variability within the E6 ORF and allowed the detection of amino acid exchanges unique for isolates from carcinomas
Murine Cytomegalovirus DNA in Peripheral Blood of Latently Infected Mice Is Detectable Only in Monocytes and Polymorphonuclear Leukocytes

AbstractCytomegalovirus (CMV), as do other herpesviruses, establishes a lifelong latent infection in its natural host. While in immunologically intact hosts most CMV infections are subclinical, clinical disease follows severe immunosuppression and immunodeficiency. In these situations CMV may produce serious life-threatening disease, and virus reactivated from the latent state is often responsible. Essential to understanding this virus and its pathogenesis is the need to define particular tissue and cell types harboring viral DNA. We searched for viral DNA and RNA in subpopulations of blood cells from mice latently infected with murine CMV by using differential centrifugation and fluorescent antibody cell sorting followed by polymerase chain reaction analysis. Following intravenous inoculation, the viral DNA was found to be present in the buffy coat at and after 21 days postinfection, and both granulocytes and peripheral blood mononuclear leukocytes (PBML) were reservoirs. Further analysis of the PBML fraction by separation into Mac-1+and Mac-1−cells revealed that monocytes harbored the DNA while lymphocytes were not sites of persistence. We conclude that in buffy coat of latently infected mice the viral DNA is present only in cells of the myeloid lineage. The relationship of this DNA to the latent infection is discussed
Identification of Sequence Changes Responsible for the Attenuation of Highly Virulent Infectious Bursal Disease Virus

AbstractThe genetic changes responsible for the attenuation of infectious bursal disease virus (IBDV) have not been defined at the molecular level, although passage of the virus in cell culture results in the loss of virulence. To understand the molecular basis of IBDV virulence and attenuation, the IBDV genome segment encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) of a cell culture-adapted OKYMT strain derived from highly virulent OKYM was cloned as cDNA, and the nucleotide sequence was determined. Comparison of the identified nucleotide and deduced amino acid sequences of the attenuated strain with the parental virulent OKYM strain revealed only five amino acid differences: four in the VP2 variable domain and one in the VP3. Two amino acid substitutions at positions 279 (Asp → Asn) and 284 (Ala → Thr) in the VP2 variable domain were commonly predicted in another cell culture-adapted strain. These two amino acid changes resulted in reduced hydrophilia of this region and deletion of the α-helix which might alter the conformation of the virion surface structures. These results may imply that the amino acid residues at position 279 and 284 in VP2 variable domain contribute to virulence of IBDV
Gene Defects Clustered at the C-Terminus of the vpr Gene of HIV-1 in Long-Term Nonprogressing Mother and Child Pair:In VivoEvolution of vpr Quasispecies in Blood and Plasma

AbstractEarlier studies on HIV-1 strains from HIV-1-infected long-term nonprogressors (LTNP) have reported that nef deletions and/or attenuations may be crucial in the survival of these patients. Other reports have suggested that the nef gene may not be the only gene involved, but attenuations in other accessory genes (vif, vpr, vpu), which play an important role in the viral life cycle, may be similarly important in chronic HIV-1 infection in LTNPs. Here we show the molecular and phylogenetic analyses of the vpr gene in HIV-1 strains derived from both blood and plasma of an HIV-1 infected long-surviving mother–child pair which has survived for >13 years with HIV infection: both have maintained stable CD4+T-cell counts. Analyses of blood- and plasma-derived HIV-1 vpr clones indicated the presence of defects (insertions and deletions) and length polymorphisms. Interestingly, all the vpr defects in PBMCs and plasma were clustered at the C-terminus of the Vpr protein, between amino acid residues 83 and 89, which has been implicated in the G2 cell cycle arrest as a step to early HIV-1 infection. In contrast, the vpr sequence analysis of HIV-1 strains derived from 30 different patients, who either died of AIDS-related illnesses or have AIDS, showed neither C-terminal defects nor length polymorphism in the vpr gene. Also, secondary structure predictions suggest that the naturally occurring mutations at the C-terminal region (aa 83–89) have the potential to affect the secondary structure of the Vpr protein. Also, in some cases, the out-of-frame mutations and the length polymorphisms affect the tat gene reading frame. Together, these mutations may have potential significance in conferring chronic HIV-1 infection in this long-surviving nonprogressing mother–child pair
Carboxyl Terminus of Bovine Papillomavirus Type-1 L1 Protein Is Not Required for Capsid Formation

AbstractThe papillomavirus major capsid protein L1 can assemble into capsidsin vitro.To identify areas within the bovine papillomavirus type-1 L1 (BPV L1) protein that are important for virus assembly, we constructed a set of 24 baculovirus recombinants expressing BPV L1 deletion mutants that span the entire L1 open reading frame. Virus-like particle (VLP) formation of the L1 mutants was examined by electron microscopy. Wild-type (wt) BPV L1 expressed in recombinant baculovirus formed VLPs, while capsomeres and aggregates were seen for most of the mutants screened. However, the C-terminal truncation mutant, lacking the last 24 amino acids (ΔC2), was observed to form VLPs (threefold more efficiently than wt BPV L1). This suggests that this C-terminal region of L1 protein is not critical for capsid formation. As capsids assembled from BPV L1 are able to agglutinate mouse red blood cells (RBC) by binding to a membrane protein, we tested the ability of the mutants to hemagglutinate mouse RBCs. Most aberrant capsids or aggregates derived from deletion mutants were unable to agglutinate the RBCs with the exception of deletion mutants Δ11 (aa 231–271), Δ14 (aa 291–331), Δ21 (aa 431–471), and the carboxyl-terminus truncation mutant ΔC2
Sequence Heterogeneity of Nef Transcripts in HIV-1-Infected Subjects at Different Stages of Disease

AbstractNeftranscripts were analyzed from peripheral blood mononuclear cells of 10 HIV-1-infected subjects with 9-822 CD4+ lymphocytes/cu mm, including 4 individuals with a probable common source infection. There was no relationship between the phylogenetic position of the variousnefsequences and the disease state of the person from whom they were derived. Thenefopen reading frame was disrupted in all three clones from only 1 subject. Functional analyses of a representative clone from each of the remaining 9 subjects showed that allnefalleles were capable of CD4 cell surface down-regulation, but only threenefalleles suppressed the induction of IL-2 transcription
The E5 Gene of HPV-16 Enhances Keratinocyte Immortalization by Full-Length DNA

AbstractHuman papillomavirus type 16 (HPV-16) is the most common papillomavirus genome found in human cervical cancers and its DNA is capable of immortalizing primary keratinocytesin vitro.When expressed by their native promoter, two separate HPV-16 oncogenes, E6 and E7, cooperate to immortalize primary human keratinocytes. Early HPV cervical lesions express abundant amounts of E5-specific RNA, and using a quantitative keratinocyte immortalization assay, we demonstrate here that E5 can actin cisto increase (4–10 fold) the efficiency of cellular immortalization by E6/E7. These results suggest that the expression of the HPV-16 E5 gene may play a role in the pathogenesis of early HPV infections by potentiating the effects of E6/E7
Genetic Organization and Diversity of the 3′ Noncoding Region of the Hepatitis C Virus Genome

AbstractThe 3′ noncoding region (3′ NCR) of the hepatitis C virus (HCV) genome contained in viral particles was analyzed by an RNA linker ligation followed by reverse transcription-polymerase chain reaction. Sequence analysis of the amplified fragment from four strains, including different genotypes 1b, 2b, 3a, and 3b indicated that the 3′ NCR is composed of between 200 and 235 nts. The sequence of the 3′ NCR consists of a type-specific region (immediately following the termination codon), a poly(U) stretch, a C(U)n-repeat, and highly conserved region termed the core element. The poly(U) stretch and C(U)n-repeat regions varied in length and in sequence among different genotypes. Core elements having putative secondary structure consisted of 98 or 100 nts and were highly conserved in all genotypes. Most of the nt changes found in different genotypes did not affect the secondary structure of the core elements, suggesting that this region may play an important role in replication, stabilization of the HCV RNA, and/or packaging of the genome. Most of the HCV-1b strains carried two U residues at the 3′ end of the core element, while the minor HCV-1b strains had no U residues, demonstrating that there are two variants in type 1b strains. Amplification of the core element using linker-primed cDNA was comparable with that using the 3′ proximal core element-primed cDNA, indicating that the 3′ end of HCV genome was terminated by an OH group