bluuebunny/crossref_metadata_2025 huggingface dataset:
Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection
Authors: Kilani R T , Chang L J , Garcia-Lloret M I , Hemmings D , Winkler-Lowen B , Guilbert L J
Published: January 2020

Whether cell-free human immunodeficiency virus type 1 (HIV-1) can productively infect placental trophoblasts (which in turn could transmit the virus into the fetal circulation) is controversial but essential to know for the evaluation of alternative routes (such as cell-mediated infection or trophoblast damage). We have addressed infection factors such as cell purity, source, culture methods, and activation states as well as virus variant and detection methods to conclusively determine the outcome of trophoblast challenge by free virus. Pure (> 99.98%) populations of trophoblasts from 11 different placentas were challenged at a multiplicity of infection (MOI) as high as 6 with five different HIV-1 variants, three of which are non-syncytium-forming, macrophage-tropic isolates from infected infants, with and without coinfection with cytomegalovirus; these preparations were monitored for productive infection for up to 3 weeks after challenge by five different criteria, the most sensitive of which were cocultivation with target cells that can detect virus at an MOI of 10(-7) and HIV DNA PCR that detects 30 virus copies per 10(5) cells. Infection was never detected. However, molecularly cloned T-cell (pNL4-3)- and macrophage (pNLAD8)-tropic provirus plasmids, when transfected into primary trophoblasts, yielded productive infections, indicating that trophoblasts do not suppress late-stage virus replication and assembly. Because of the purity of the trophoblast preparations, the extended length of the infection culture period, the number of trophoblast preparations and virus types examined, the sensitivity of the bioassays and molecular detection assays, and the observations that trophoblasts can support virus replication from provirus, the results of this study strongly argue that free virus cannot infect primary villous trophoblasts.
Phosphorylation sites in polyomavirus large T antigen that regulate its function in viral, but not cellular, DNA synthesis
Authors: Chatterjee A , Bockus B J , Gjørup O V , Schaffhausen B S
Published: January 2020

Polyomavirus large T antigen (large T) is a highly phosphorylated protein that can be separated by proteolysis into two domains that have independent function. A cluster of phosphorylation sites was found in the protease-sensitive region connecting the N-terminal and C-terminal domains. Edman degradation of 32P-labeled protein identified serines 267, 271, and 274 and threonine 278 as sites of phosphorylation. Analysis of site-directed mutants confirmed directly that residues 271, 274, and 278 were phosphorylated. Threonine 278, shown here to be phosphorylated by cyclin/cyclin-dependent kinase activity, is required for viral DNA replication in either the full-length large T or C-terminal domain context. The serine phosphorylations are unimportant in the C-terminal domain context even though their mutations activates viral DNA replication in full-length large T. This finding suggests that these sites may function in relating the two domains to each other. Although the phosphorylation sites were involved in viral DNA replication, none was important for the ability of large T to drive cellular DNA replication as measured by bromodeoxyuridine incorporation, and they did not affect large T interactions with the Rb tumor suppressor family.
Drug resistance during indinavir therapy is caused by mutations in the protease gene and in its Gag substrate cleavage sites
Authors: Zhang Y M , Imamichi H , Imamichi T , Lane H C , Falloon J , Vasudevachari M B , Salzman N P
Published: January 2020

Two different responses to the therapy were observed in a group of patients receiving the protease inhibitor indinavir. In one, suppression of virus replication occurred and has persisted for 90 weeks (bDNA, < 500 human immunodeficiency virus type 1 [HIV-1] RNA copies/ml). In the second group, a rebound in virus levels in plasma followed the initial sharp decline observed at the start of therapy. This was associated with the emergence of drug-resistant variants. Sequence analysis of the protease gene during the course of therapy revealed that in this second group there was a sequential acquisition of protease mutations at amino acids 46, 82, 54, 71, 89, and 90. In the six patients in this group, there was also an identical mutation in the gag p7/p1 gag protease cleavage site. In three of the patients, this change was seen as early as 6 to 10 weeks after the start of therapy. In one patient, a second mutation occurred at the gag p1/p6 cleavage site, but it appeared 18 weeks after the time of appearance of the p7/p1 mutation. Recombinant HIV-1 variants containing two or three mutations in the protease gene were constructed either with mutations at the p7/p1 cleavage site or with wild-type (WT) gag sequences. When recombinant HIV-1-containing protease mutations at 46 and 82 was grown in MT2 cells, there was a 68% reduction in its rate of replication compared to the WT virus. Introduction of an additional mutation at the gag p7/p1 protease cleavage site compensated for the partially defective protease gene. Similarly, rates of replication of viruses with mutations M46L/I, I54V, and V82A in protease were enhanced both in the presence and in the absence of Indinavir when combined with mutations in the gag p7/p1 and the gag p1/p6 cleavage sites. Optimal rates of virus replication require protease cleavage of precursor polyproteins. A mutation in the cleavage site that enhanced the availability of a protein that was rate limiting for virus maturation would confer on that virus a significant growth advantage and may explain the uniform emergence of viruses with alterations at the p7/p1 cleavage site. This is the first report of the emergence of mutations in the gag p7/p1 protease cleavage sites in patients receiving protease therapy and identifies this change as an important determinant of HIV-1 resistance to protease inhibitors in patient populations.
Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species
Authors: Bruschke C J , Hulst M M , Moormann R J , van Rijn P A , van Oirschot J T
Published: January 2020

Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses.
The latency-associated promoter of herpes simplex virus type 1 requires a region downstream of the transcription start site for long-term expression during latency
Authors: Lokensgard J R , Berthomme H , Feldman L T
Published: January 2020

The latency-associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1) is unique among the many promoters on the viral genome in that it remains active during the latent state. We have previously shown that a DNA fragment comprising the LAT promoter element through the cap site, when moved from the LAT locus to the glycoprotein C gene, is capable of only short-term expression. These and other data suggested that an HSV DNA element from the repeat region, not included in the LAT promoter itself, might be needed to preserve long-term expression. Based on a number of recombinant viruses, we narrowed our search for this putative element to a region 3' of the LAT transcription start site. In the present study, we have shown that a 1.1-kb DNA fragment containing the putative long-term expression element (LTE) is able to restore latent-phase gene expression to the LAT promoter. The element appeared to function best when it was placed in its natural location, which is 3' of the LAT promoter; however, partial function was obtained when the LTE was inserted upstream of the LAT promoter in the reverse direction. These data indicate that the LAT promoter region is more complex than originally anticipated and that in addition to requiring both core promoter and neuronal transcription factor binding sites, the promoter requires a specific region of DNA to prevent its shutoff during a latent infection.
The herpes simplex virus type 1 immediate-early protein ICP0 is necessary for the efficient establishment of latent infection
Authors: Wilcox C L , Smith R L , Everett R D , Mysofski D
Published: January 2020

The immediate-early protein ICP0 of herpes simplex virus type 1 (HSV-1) is not essential for viral replication. However, ICP0 is important for efficient viral replication during the productive infection and for reactivation of latent HSV-1 in vivo. The in vitro model of HSV-1 latency in dorsal root ganglia neurons was used to examine the role of ICP0 in the individual steps that could lead to the appearance of a decreased reactivation phenotype of ICP0 mutant viruses. After establishment of latent infections in the neuronal cultures, induction of reactivation by nerve growth factor (NGF) deprivation resulted in the production of infectious virus with delayed kinetics and a burst size that was significantly decreased for the ICP0 mutants compared with wild-type HSV-1. The efficiency of establishment of latency with the ICP0 mutants was similarly decreased at least 10-fold, as measured by three criteria: (i) the percentage of neurons expressing the major latency-associated transcript during the latent infection, (ii) the amount of viral DNA detected in the neuronal cultures, and (iii) the percentage of neurons expressing ICP4 immunoreactivity after the induction of reactivation. The most striking finding was that ICP0 supplied by an adenovirus vector significantly restored the ability of an ICP0 mutant to establish latency and reactivation. These results strongly indicate a critical role for ICP0 in the establishment of the latent HSV-1 infection in the in vitro neuronal model.
Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs
Authors: Walker S L , Wonderling R S , Owens R A
Published: January 2020

The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity.
Herpes simplex virus genome replication and transcription during induced reactivation in the rabbit eye
Authors: Devi-Rao G B , Aguilar J S , Rice M K , Garza H H , Bloom D C , Hill J M , Wagner E K
Published: January 2020

PCR analysis of herpes simplex virus (HSV) genome replication and productive-cycle transcription was used to examine the role of the cornea in the latency-associated transcript (LAT)-mediated reactivation of HSV type 1 (HSV-1) in the rabbit eye model. The reduced relative reactivation frequency of 17 delta Pst (a LAT- virus) compared to those of wild-type and LAT+ rescuants correlated with reduced levels of viral DNA and transcription in the cornea following epinephrine induction. The timing of virus appearance in the cornea was most consistent with tissue peripheral to the cornea itself mediating a LAT-sensitive step in the reactivation process. Specific results include the following. (i) While viral DNA was found in the corneas of rabbits latently infected with either the LAT+ or LAT- virus prior to and during the first 16 to 24 h following induction, more was found in animals infected with the LAT+ virus. (ii) A significant increase in levels of viral DNA occurred 20 to 168 h following induction. (iii) The average relative amount of viral DNA was lower at all time points following reactivation of animals infected with the LAT- virus. (iv) Expression of productive-cycle transcripts could be detected in corneas of some rabbits latently infected with either the LAT+ or LAT- virus, and the amount recovered and the timing of appearance differed during the reactivation of rabbits latently infected with the LAT+ or LAT- virus. (v) Despite the reduced recoveries of LAT- virus DNA and productive-cycle transcripts in reactivating corneas in vivo compared to those of their LAT+ counterparts, such differences were not detected in cultured keratinocytes or in experiments in which relatively high titers of virus were superinfected into the eyes of latently infected rabbits. (vi) A number of LAT(+)-virus-infected rabbits expressed LAT in corneas isolated from uninduced rabbits. When seen, its amount was significantly higher than that of a productive-cycle (VP5) transcript.
Infectious particles derived from Semliki Forest virus vectors encoding murine leukemia virus envelopes
Authors: Lebedeva I , Fujita K , Nihrane A , Silver J
Published: January 2020

Semliki Forest virus vectors encoding murine leukemia virus (MLV) envelope protein with a truncated cytoplasmic tail generate submicrometer, cell-associated, membranous particles that transmit replication-competent vector RNA specifically to cells bearing the MLV receptor. Such "minimal" viruses could have applications as retroviral vaccines or in the study of virus evolution.
The relationship between tumor necrosis factor and human immunodeficiency virus gene expression in lymphoid tissue
Authors: Li Q , Gebhard K , Schacker T , Henry K , Haase A T
Published: January 2020

In tissue culture models of chronic human immunodeficiency virus type 1 (HIV-1) infection, cytokines such as tumor necrosis factor alpha (TNF-alpha) activate viral gene expression. We sought evidence that TNF-alpha might similarly regulate viral gene expression in vivo in the major lymphoid tissue (LT) reservoir. We used in situ hybridization, quantitative image analysis, and double-label techniques to compare cytokine and HIV-1 RNA levels in sections of tonsil and lymph node tissues obtained from individuals in early and later stages of HIV-1 infection. The levels of TNF-alpha gene expression in LT from HIV-1-infected an uninfected individuals were indistinguishable, and we found no correlation between TNF-alpha gene expression in LT and the level of HIV-1 gene expression in LT. There is thus little evidence that in vivo TNF-alpha significantly influences HIV production in LT.
Active-site residues of cyclophilin A are crucial for its incorporation into human immunodeficiency virus type 1 virions
Authors: Dorfman T , Weimann A , Borsetti A , Walsh C T , Göttlinger H G
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) incorporates the cellular peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). CsA inhibits the incorporation of CyPA and reduces HIV-1 virion infectivity but is inactive against closely related primate lentiviruses that do not interact with CyPA. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction with a proline-containing, solvent-exposed loop in the capsid (CA) domain of the Gag polyprotein. CsA, which disrupts the interaction with CA, binds at the active site of CyPA. To test whether active-site residues are also involved in the interaction with HIV-1 CA, we used a panel of previously characterized active-site mutants of human CyPA. Expression vectors for epitope-tagged wild-type and mutant CyPA were transfected into COS-gamma cells along with HIV-1 proviral DNA, and the virions produced were analyzed for the presence of tagged proteins. Cotransfection of the wild-type expression vector led to the incorporation of readily detectable amounts of epitope-tagged CyPA into HIV-1 virions. One CyPA mutant with a substantially decreased sensitivity to CsA was incorporated with wild-type efficiency, demonstrating that the requirements for binding to CsA and to HIV-1 CA are not identical. The remaining six CyPA mutants were incorporated with markedly reduced efficiency, providing in vivo evidence that HIV-1 CA interacts with the active site of CyPA.
Requirement of Protein Synthesis for Bacteriophage φX174 Superinfection Exclusion
Authors: Hutchison Clyde A. , Sinsheimer Robert L.
Published: January 2020

Within 5 to 10 min at 37 C, bacteria infected by bacteriophage φX174 acquire the ability to exclude superinfecting φX particles from reproducing themselves. The superinfecting phage are blocked at a stage prior to synthesis of the parental replicative form molecule; the superinfecting deoxyribonucleic acid remains as intact (infective) single strands. Establishment of superinfection exclusion and its maintenance require protein synthesis.
Synthesis of Virus-Specific Ribonucleic Acid in KB Cells Infected with Type 2 Adenovirus
Authors: Lucas Joseph J. , Ginsberg Harold S.
Published: January 2020

By using the technique of deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybridization, virus-specific RNA (cRNA) was detected 6 hr after infection in preparations of total RNA from cells infected with type 2 adenovirus in the presence of 2 μ
m
5-fluorodeoxyuridine. In the absence of 5-fluorodeoxyuridine, there was a continuous increase in the incorporation of
3
H-uridine into viral cRNA until 20 hr after infection, at which time approximately 40% of the
3
H-uridine entering RNA was found in virus-specific RNA. When RNA was prepared from polyribosome fractions obtained from cytoplasmic extracts of infected cells, virus-directed transcription was detected at 3 hr after infection (i.e., 3 to 4 hr before the initiation of viral DNA synthesis). Viral cRNA species synthesized at different times after infection were compared by the technique of DNA-RNA hybridization-inhibition (“presaturation” hybridization-competition). Three hybridization-inhibition techniques were compared. The techniques differed in the manner in which the DNA-RNA complex was isolated after the first hybridization reaction. Depending on the procedure employed, various degrees of inhibition were measured. The variation could be essentially eliminated if prior to hybridization the inhibitory RNA species were alkali-degraded to a uniform size of about 4
S
. Undegraded RNA could be used if the DNA-RNA complex was isolated by using a procedure involving rigorous washing (preferably including ribonuclease treatment) before the second hybridization with labeled RNA. When a rigorous hybridization-inhibition procedure was used, three classes of virus-specific RNA species could be distinguished: (i) early RNA class I whose synthesis began prior to viral DNA replication and stopped at some time after the initiation of viral DNA replication—it comprised about 70% of the early RNA species and was apparently degraded by 18 hr after infection; (ii) early RNA class II whose synthesis began prior to viral DNA replication and apparently continued at an enhanced rate late in infection; and (iii) late RNA whose synthesis began after the initiation of viral DNA synthesis.
Bacteriophage Particles with Endo-Glycosidase Activity
Authors: Stirm Stephan , Bessler Wolfgang , Fehmel Frank , Freund-Mölbert Elisabeth
Published: January 2020

Some
Escherichia coli
K bacteriophage particles, capable of interacting specifically with bacterial polysaccharide capsules, carry an endo-glycosidase activity, probably located in the spikes.
Role of Genes 46 and 47 in Bacteriophage T4 Reproduction
Authors: Hosoda Junko , Mathews Elaine , Jansen Barbara
Published: January 2020

Functional proteins coded by genes 46 and 47 are required for (i) continuation of deoxyribonucleic acid (DNA) synthesis in the late period of T4 infection and (ii) production of normal, late replicating DNA which contains strands with a sedimentation coefficient in alkaline sucrose greater than that of mature DNA (73
S
). Continued DNA synthesis in the late period in the absence of functional genes 46 or 47 can be achieved by inhibiting late protein synthesis either by using bacterio-phage with a second mutation in gene 55 or by adding chloramphenicol to the culture before the decline in the rate of DNA synthesis. However, when functional 46/47 proteins are absent throughout infection, no strands with a sedimentation coefficient greater than 73
S
(in alkaline sucrose) are produced. This is the case even when DNA synthesis is allowed to continue. DNA arrest is accompanied by conversion of rapidly sedimenting, replicating DNA to slower sedimenting forms. When 46/47 is absent from the beginning of infection, the conversion product has a smaller sedimentation coefficient than mature DNA both in neutral and alkaline sucrose. When DNA arrest occurs midway in infection by heat-inactivating the ts46 enzyme, the conversion product has a sedimentation coefficient (i) the same as mature DNA in both neutral (63
S
) and alkaline sucrose if capsid assembly is allowed to take place and (ii) close to 63
S
in neutral sucrose but heterogenous and relatively greater (up to 100
S
) in alkaline sucrose if capsid assembly is inhibited. The structure of this DNA is unknown.
Relationship Between the Ribonucleic Acid-Synthesizing Capacity of Ultraviolet-Irradiated Newcastle Disease Virus and Its Ability to Induce Interferon
Authors: Clavell Luis A. , Bratt Michael A.
Published: January 2020

Ultraviolet (UV)-irradiated Newcastle disease virus which has lost its infectivity but has the capacity to induce interferon also has the capacity to induce ribonucleic acid (RNA) synthesis both in vitro and early in infection in vivo. With large doses of UV irradiation, RNA-synthesizing capacity and interferon-inducing capacity are lost in parallel. Limited amounts of base-paired RNA associated with a transcriptive intermediate are involved in this RNA synthesis. These findings suggest the possibility that the single-stranded RNA of the UV-irradiated virus induces interferon by serving as a template for the synthesis of base-paired RNA. UV irradiation of the virus breaks down viral RNA but at a rate which is too slow to be a major cause of the loss of RNA-synthesizing capacity. Evidence is presented which suggests that less of the template RNA of the UV-irradiated virus is copied and that the product which is synthesized is smaller than that synthesized by nonirradiated virus.
Mechanism of Intranuclear Crystal Formation of Herpes Simplex Virus as Revealed by the Negative Staining of Thin Sections
Authors: Miyamoto Kaneatsu
Published: January 2020

Structural alterations induced in HeLa cells by herpes simplex virus and the mechanism whereby the virus is formed in the nucleus in crystal arrays were studied by electron microscopy with both the usual and negatively stained sections. Aggregates of granular and filamentous material were observed in the cytoplasm of infected cells with both sections. On the other hand, no remarkable alterations in appearance of the cytoplasmic ground substance were observed with the usual sections of infected cells. However, the cytoplasmic ground substance of infected cells when negatively stained consisted of granular material which was different in appearance from the spongy material constituting the cytoplasmic matrix of uninfected cells. In the nucleus of infected cells, complexes consisting of round bodies, amorphous material, aggregates of uniform granules in rows, and viral crystals were often observed near the nuclear membrane in both types of sections. Examinations of the granular aggregates with negatively stained sections suggested that each granule represents a subunit and that the several adjoining subunits (approximately eight) constitute the requirement for formation of a single viral capsid with a core. Thus, rapid and simultaneous formation of the core and capsid within the aggregate would replace the rows of the granules with the viral crystal. The advantages of negative staining of thin sections for visualization of fine structural alterations are discussed.
Antigenic Specificity of Poliovirus-Related Particles
Authors: Ghendon Y. Z. , Yakobson E. A.
Published: January 2020

Poliovirus-specific structures with sedimentation constants of 5, 14, 73, and 150
S
produced in infected HeLa cells possess different antigenic specificities.
Specific Suppression of Mutations in Genes 46 and 47 by
das
, a New Class of Mutations in Bacteriophage T4D
Authors: Hercules Kitty , Wiberg John S.
Published: January 2020

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis (“DNA arrest”) and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named
das
for “
D
NA
a
rrest
s
uppressor.” The
das
mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on
Escherichia coli
B, whereas [46, 47] mutants make none. The [
das
, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [
das
, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the
das
mutation appears to be gene-specific: it suppresses both
amber
and temperature-sensitive mutations in genes 46 and 47 and does not suppress
amber
mutations in any of the other genes tested. The [
das
] single mutants make normal-sized plaques on
E. coli
B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The
das
mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.
Inhibition of the Synthesis of Simian Virus 40 Antigens in Cells Preinfected with Yaba Tumor Virus
Authors: Tsuchiya Y. , Rouhandeh H.
Published: January 2020

The synthesis of tumor and viral antigens after infection of an established line of cynomolgus monkey kidney cells with simian virus 40 (SV40) was compared in cells previously infected with Yaba virus and in cells not preinfected. SV40 failed to induce synthesis of tumor or viral antigens in cells preinfected with Yaba virus. The inhibitory state in preinfected cells was shown to develop sequentially. Increase in the rate of deoxyribonucleic acid synthesis in the nuclei of preinfected cells occurred after infection with SV40. This rate of increase was significantly lower than that which occurred in SV40-infected cells which had not been preinfected. Cytosine arabinoside did not exert significant effect on the development of the inhibitory effect against SV40 in Yaba virus-infected cells.
Inactivation of Nocardiophages φC and φEC by Extracts of Bacteriophage-Attachable Cells
Authors: Brownell George H. , Crockett Jennifer K.
Published: January 2020

Cultures of several species of
Nocardia
, including
N. erythropolis
Mat-Ce and Mat-cE mating strains, were extracted with solvents in an attempt to isolate an inactivating complex for nocardiophages φC and φEC. Ethanol was the only solvent found effective in solubilizing an inhibitory substance. Inactivating extracts were obtained from the cells of all species to which the phage were able to attach. After extraction of whole cells or cell wall preparations, the phage could not effectively attach to them. Both phages φC and φEC were inactivated by the same complex. However, phage φEC inactivation was 10-fold greater than φC inactivation. The velocity of inactivation was about 4.1 × 10
2
plaque-forming units per microgram per minute for φC and 1.1 × 10
3
plaque-forming units per microgram per minute for phage φEC. The cell extracts required divalent cations for phage inactivation. The inhibitory capacity of the cell extracts was reduced or lost by the activity of proteolytic enzymes, Tween 80, 2-mercaptoethanol, thymol, and sodium lauryl sulfate. Boiling the extract for 10 min did not alter its activity. The inactivating substance was postulated to be a lipoprotein of considerable complexity, unique in the ease with which it is solubilized from host cells by ethanol.
Polyamines and the Delay in Deoxyribonucleic Acid Synthesis in Some Bacteriophage T4 Infections
Authors: Dion Arnold S. , Cohen Seymour S.
Published: January 2020

Escherichia coli
B infected by the DD mutant of T4,
am
N116, is stimulated to initiate deoxyribonucleic acid accumulation by 1 to 10 m
m
spermidine but not by 10 m
m
putrescine. The syntheses of putrescine and spermidine in cells infected by T4D and the mutant are similar, although slight differences are observed in the intracellular concentration of free spermidine. Unlike r-K12 (λ) systems,
am
N116-infected cells do not leak polyamine.
Genetic Location of a Mutant of Bacteriophage T4 Deficient in the Ability to Induce Endonuclease II
Authors: Ray Purnima , Sinha Navin K. , Warner Huber R. , Snustad D. Peter
Published: January 2020

Reciprocal three-factor crosses and the use of a partial revertant of a putative ribonucleotide reductase mutant of
Escherichia coli
B/5 as indicator have made it possible to map
denA
(deficient in endonuclease II) between
nrd-11
(ribonucleotide reductase gene B) and
amM69
(gene 63) on the bacteriophage T4 chromosome.
Proteins Specified by Herpes Simplex Virus VI. Viral Proteins in the Plasma Membrane
Authors: Heine Jochen W. , Spear Patricia G. , Roizman Bernard
Published: January 2020

Previous papers in the series have shown that the surface membranes of herpesvirus-infected cells acquire new immunological specificities and that purified infected cell membrane preparations, characterized by their physical properties rather than topology in the cell, contain new glycoproteins genetically determined by the virus. In this study, we prepared purified plasma membrane identified by its 5′ nucleotidase, fucose, and reduced nicotinamide adenine dinucleotide-diaphorase content. Analysis of the membrane proteins and glycoproteins by electrophoresis in acrylamide gels indicated the following. (i) Purified plasma membranes from infected cells contained two sets of proteins, i.e., host proteins were present both before and after infection and viral proteins were present only after infection. (ii) After infection, no appreciable selective or nonselective loss of host proteins from membranes was demonstrable. However, no new host proteins were made. (iii) Electropherograms of plasma membrane proteins from infected cells indicated the presence of at least 12 virus-specific proteins ranging in molecular weight from 25 × 10
3
to 126 × 10
3
daltons. Of these, at least nine were glycosylated. Proteins and glycoproteins with similar electrophoretic mobilities but in somewhat different ratios were also present in preparations of highly purified virions.
Relationship Between
Escherichia coli
B Titer and the Level of Deoxycytidylate Deaminase Activity Induced on Bacteriophage T2r
+
Infection
Authors: Trimble Robert B. , Maley Gladys F. , Maley Frank
Published: January 2020

The activities of six bacteriophage T2r
+
-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected
Escherichia coli
B from 8 × 10
8
to 2 × 10
8
cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 × 10
8
to 8 × 10
8
per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen.
Virus-Associated Nucleases: Evidence for Endonuclease and Exonuclease Activity in Rabbitpox and Vaccinia Viruses
Authors: Aubertin Anne-Marie , McAuslan B. R.
Published: January 2020

The endonucleolytic action of a deoxyribonuclease activity in rabbitpox and vaccinia virus was established by change in sedimentation rate of denatured
3
H-lambda deoxyribonucleic acid substrate. The presence of two deoxyribonuclease activities in pox-virus is confirmed. Exo- and endonuclease activities are unmasked by treatment of purified virus with the detergent Nonidet P-40 and further enhanced by treatment of viral “cores” with trypsin.
Simple Method for the Isolation of Encephalomyocarditis Virus Ribonucleic Acid
Authors: Kerr I. M. , Martin E. M.
Published: January 2020

A simple purification procedure for encephalomyocarditis virus is described, yielding material suitable for extraction of viral ribonucleic acid for use as messenger in animal cell-free systems.
Specific Endonuclease R Fragments of Bacteriophage φX174 Deoxyribonucleic Acid
Authors: Edgell Marshall H. , Hutchison Clyde A. , Sclair Morton
Published: January 2020

The restriction enzyme from
Hemophilus influenzae,
endonuclease R, cleaves φX174 replicative-form deoxyribonucleic acid (DNA) into at least 13 specific limit fragments. The molecular weights of 12 of the fragments have been estimated by gel electrophoresis and electron microscopy. Using the genetic assay for small fragments of φX DNA, we have shown that we can salvage markers from the endonuclease R φX-RF fragments.
Isolation of Bacteriophages from Group H Streptococci
Authors: Parsons C. Lowell , Colon Alba E. , Leonard Carmen G. , Cole Roger M.
Published: January 2020

Temperate phages were isolated from 5 of the 17 strains of group H streptococci tested.
Absence of Leukosis Virus Markers in Hamster Cells Transformed by Herpes Simplex Virus Type 2
Authors: Rapp Fred , Conner Richard , Glaser Ronald , Duff Ronald
Published: January 2020

Hamster cells which transformed after exposure to herpes simplex virus type 2 previously irradiated with ultraviolet light were tested for the presence of hamster leukosis viruses or their virus-specific antigens. Three techniques that were used did not detect the presence of either hamster C-type particles or antigens commonly associated with such structures. The implication of these findings to current concepts of virus oncology is discussed.
Assay for Secondary Structure in Ribonucleic Acid
Authors: Engelhardt Dean L.
Published: January 2020

A chromatographic procedure is described which can discriminate among single-stranded ribonucleic acid (RNA) molecules in solution on the basis of the extent of their secondary structure. The assay is effected through chromatography at different temperatures with columns of cellulose CF-11. When Sindbis virus RNA is chromatographed in this system, the ratio of the amounts of RNA eluting in the single-stranded peak to those eluting in the double-stranded peak increases at higher temperatures, presumably a measure of the relative amounts of Sindbis virus RNA secondary structure at different temperatures. With this assay, Sindbis virus RNA, phage f2 RNA, and polyuridylate have been found to have different amounts of secondary structure.
Computational and Experimental Investigation of Biofilm Disruption Dynamics Induced by High-Velocity Gas Jet Impingement
Authors: Prades Lledó , Fabbri Stefania , Dorado Antonio D. , Gamisans Xavier , Stoodley Paul , Picioreanu Cristian
Published: January 2020

Knowledge of mechanisms promoting disruption though mechanical forces is essential in optimizing biofilm control strategies which rely on fluid shear. Our results provide insight into how biofilm disruption dynamics is governed by applied forces and fluid properties, revealing a mechanism for ripple formation and fluid-biofilm mixing. These findings have important implications for the rational design of new biofilm cleaning strategies with fluid jets, such as determining optimal parameters (e.g., jet velocity and position) to remove the biofilm from a certain zone (e.g., in dental hygiene or debridement of surgical site infections) or using antimicrobial agents which could increase the interfacial area available for exchange, as well as causing internal mixing within the biofilm matrix, thus disrupting the localized microenvironment which is associated with antimicrobial tolerance. The developed model also has potential application in predicting drag and pressure drop caused by biofilms on bioreactor, pipeline, and ship hull surfaces.
Development of Candida auris Short Tandem Repeat Typing and Its Application to a Global Collection of Isolates
Authors: de Groot Theun , Puts Ynze , Berrio Indira , Chowdhary Anuradha , Meis Jacques F.
Published: January 2020

Candida auris
is an emerging fungal pathogen now recognized as a threat to public health. The pathogen has spread worldwide and causes mainly hospital-associated outbreaks. To track and trace outbreaks and to relate them to new introductions from elsewhere, whole-genome sequencing and amplified fragment length polymorphism (AFLP) have been used for molecular typing. Whole-genome sequencing is costly and available only at a few centers, and AFLP is a complicated technique and hard to interpret. We describe a novel simple STR genotyping technique based on short tandem repeats in the
C. auris
genome. We also show that the performance of this STR-based genotyping technique has proven comparable to that of WGS. Overall, this work provides a novel, rapid, reliable, and cost-effective method of molecular outbreak investigations of
C. auris
.
Plasmodium yoelii Erythrocyte-Binding-like Protein Modulates Host Cell Membrane Structure, Immunity, and Disease Severity
Authors: Peng Yu-chih , Qi Yanwei , Zhang Cui , Yao Xiangyu , Wu Jian , Pattaradilokrat Sittiporn , Xia Lu , Tumas Keyla C. , He Xiao , Ishizaki Takahiro , Qi Chen-Feng , Holder Anthony A. , Myers Timothy G. , Long Carole A. , Kaneko Osamu , Li Jian , Su Xin-zhuan
Published: January 2020

Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC).Plasmodium yoeliiEBL (PyEBL) is known to play a role in parasite invasion of RBCs. Here, we show that PyEBL also affects disease severity through modulation of host immune responses, particularly type I interferon (IFN-I) signaling. This discovery assigns a new function to PyEBL and provides a mechanism for developing disease control strategies.
Progressive Microbial Community Networks with Incremental Organic Loading Rates Underlie Higher Anaerobic Digestion Performance
Authors: Wu Linwei , Shan Xiaoyu , Chen Si , Zhang Qiuting , Qi Qi , Qin Ziyan , Yin Huaqun , Zhou Jizhong , He Qiang , Yang Yunfeng
Published: January 2020

AD is a biological process widely used for effective waste treatment throughout the world. Biotic interactions among microbes are critical to the assembly and functioning of the microbial community, but the response of microbial interactions to environmental changes and their influence on AD performance are still poorly understood. Using well-replicated time series data of 16S rRNA gene amplicons and functional gene arrays, we constructed random matrix theory-based association networks to characterize potential microbial interactions with incremental OLRs. We demonstrated striking linkage between network topological features of methanogenic archaea and AD functioning independent of environmental parameters. As the intricate balance of multiple microbial functional groups is responsible for methane production, our results suggest that microbial interaction may be an important, previously unrecognized mechanism in determining AD performance.
Post-tonsillectomy dysphagia secondary to glossopharyngeal nerve injury
Authors: Hong Scott A , LaGorio Lisa , Husain Inna
Published: January 2020

The glossopharyngeal nerve lies in close proximity to the tonsillar fossa making it susceptible to injury during tonsillectomy, though it is uncommonly injured in practice. We present a case of severe dysphagia due to glossopharyngeal nerve injury following tonsillectomy and demonstrate the efficacy of the McNeill Dysphagia Therapy Program (MDTP), a novel stepwise exercise-based rehabilitation program, in improving clinical outcomes. In patients with persistent, severe dysphagia following tonsillectomy, glossopharyngeal nerve injury should be on the differential diagnosis. The MDTP is one method by which clinicians may be able to treat this complex condition.
Utilisation of emergency departments of behavioural disorders and supply of workforce in Nebraska
Authors: Wani Rajvi Jayant , Watanabe-Galloway Shinobu , Tak Hyo Jung , Chen Li-Wu , Wehbi Nizar , Wilson Fernando
Published: January 2020

Background

Emergency departments (EDs) have become entry points for treating behavioural health (BH) conditions, thereby rendering the evaluation of their utilisation necessary.

Objectives

This study estimated behavioural-related hospital-based ED visits and outcomes of leaving against medical advice as well as the incurred charges within the primarily rural State of Nebraska. Also, the study correlated behavioural workforce distribution and location of EDs with ED utilisation.

Methods

Nebraska State Emergency Department Database provided information on utilisation of services, charges, diagnoses and demographic. Health Professional Tracking Services survey provided the distribution of EDs and BH workforce by region. To examine the effect of patient characteristics on discharge against medical advice, multivariable logistic regression modelling was used.

Findings

US$96.4 million were ED charges for 52 035 visits for BH disorders over 3 years. Of these, 35% and 50% were between 25 and 44-years old and privately insured, respectively. The uninsured (OR:1.53, p=0.0047) and 45–64 years old (OR:2.31, p<0.001) had higher odds of leaving against medical advice. The findings from this study identified ED outcomes among high-risk cohort.

Conclusions

There were high ED rates among the limited number EDs facilities in rural Nebraska. Rural regions of Nebraska faced workforce shortages and had high numbers of ED visits at relatively few accessible EDs.

Clinical implications

Customised rural-centric public health programmes, which are based in clinical settings, can encourage patients to adhere to ED-treatment. Also, increasing the availability of BH workforce (either via telehealth or part-time presence) in rural areas can alleviate the problem and reduce ED revisits.
Untapped potential? Attitudes and behaviours of forestry employers toward the Indigenous workforce in Quebec, Canada
Authors: Proulx Guillaume , Beaudoin Jean-Michel , Asselin Hugo , Bouthillier Luc , Théberge Delphine
Published: January 2020

The skilled labour shortage in the natural resource sector is a major issue in North America, particularly in the Canadian forestry sector. In the province of Quebec alone, 15 000 positions will need to be filled by 2022. At the same time, many Indigenous communities are seeking to develop employment opportunities, as they have high unemployment rates and a young and growing population. But are forestry employers creating an environment conducive to the recruitment, integration, and retention of an Indigenous workforce? We interviewed 22 directors and human resource managers from 19 forestry businesses (16 non-Indigenous and 3 Indigenous) in Quebec, with a view to answering this question. Employer narratives suggest that they have only just begun to see the potential of the Indigenous workforce and put in place diversity management practices. Partnerships between Indigenous communities and forestry businesses, development of alternative training and skill development methods, and awareness-raising among employees and employers were found to favour recruitment, integration, and retention of Indigenous workers. Conversely, according to participants, stereotypes, discrimination, lack of inclusion measures, drug and alcohol use, and lack of training reduce the potential for Indigenous people to join the forestry workforce.
Stand density, drought, and herbivory constrain ponderosa pine regeneration pulse
Authors: Kolb Thomas E. , Flathers Kelsey , Bradford John B. , Andrews Caitlin , Asherin Lance A. , Moser W. Keith
Published: January 2020

Trees in dry forests often regenerate in episodic pulses when wet periods coincide with ample seed production. Factors leading to success or failure of regeneration pulses are poorly understood. We investigated the impacts of stand thinning on survival and growth of the 2013 cohort of ponderosa pine (Pinus ponderosa Douglas ex P. Lawson & C. Lawson) seedlings in northern Arizona, United States. We measured seedling survival and growth over the first five growing seasons after germination in six stand basal areas (BAs; 0, 7, 14, 23, 34, and 66 (unthinned) m2·ha−1) produced by long-term experimental thinnings. Five-year survival averaged 2.5% and varied among BAs. Mean survival duration was longer in intermediate BAs (11 to 16 months) than in clearings and high BAs (5 months). The BAs of 7, 14, and 23 m2·ha−1had >2600 5-year-old seedlings·ha−1. In contrast, regeneration was lower in the clearing (666 seedlings·ha−1) and failed completely in the 34 m2·ha−1and unthinned treatments. Seedling survival was highest during wet years and lowest during drought years. Many surviving seedlings had no net height growth between years 4 and 5 because of stem browsing. Results indicate that natural regeneration of ponderosa pine is influenced by stand BA, drought, herbivory, and interactions between extreme climatic events.