Number Of Infectious Centers Research Articles

Dual-Isotope SPECT Imaging with NIS Reporter Gene and Duramycin to Visualize Tumor Susceptibility to Oncolytic Virus Infection

Noninvasive dual-imaging methods that provide an early readout on tumor permissiveness to virus infection and tumor cell death could be valuable in optimizing development of oncolytic virotherapies. Here, we have used the sodium iodide symporter (NIS) and 125I radiotracer to detect infection and replicative spread of an oncolytic vesicular stomatitis virus (VSV) in VSV-susceptible (MPC-11 tumor) versus VSV-resistant (CT26 tumor) tumors in BALB/c mice. In conjunction, tumor cell death was imaged simultaneously using technetium (99mTc)-duramycin that binds phosphatidylethanolamine in apoptotic and necrotic cells. Dual-isotope single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed areas of virus infection (NIS and 125I), which overlapped well with areas of tumor cell death (99mTc-duramycin imaging) in susceptible tumors. Multiple infectious foci arose early in MPC-11 tumors, which rapidly expanded throughout the tumor parenchyma over time. There was a dose-dependent increase in numbers of infectious centers and 99mTc-duramycin-positive areas with viral dose. In contrast, NIS or duramycin signals were minimal in VSV-resistant CT26 tumors. Combinatorial use of NIS and 99mTc-duramycin SPECT imaging for simultaneous monitoring of oncolytic virotherapy (OV) spread and the presence or absence of treatment-associated cell death could be useful to guide development of combination treatment strategies to enhance therapeutic outcome.

Effects of Genotype and Isolate on Expression of Dollar Spot in Seashore Paspalum

Seashore paspalum (Paspalum vaginatum Swartz) is a warm-season turfgrass species primarily used on golf courses and athletic fields, and is often impacted by the disease dollar spot caused by Sclerotinia homoeocarpa F.T. Bennett. Dollar spot is the most common and economically important turfgrass disease in North America, and current management of this disease relies heavily on frequent fungicide applications. An alternate management strategy is host plant resistance, but a better understanding of the interactions between pathogen isolates and the host species is needed to effectively incorporate this resistance into elite seashore paspalum genotypes. The goal of this study was to gather host plant/isolate response data that could be used to develop an effective and efficient screening protocol for resistance to this important disease. Five genotypes of seashore paspalum (‘Aloha’, ‘SeaIsle 2000’, ‘SeaIsle 1’, ‘SeaIsle Supreme’, and 05-1743) varying in dollar spot resistance were inoculated with five isolates of S. homoeocarpa in repeated field studies during 2012 and 2013. Isolates used were from three warm-season and one cool-season turfgrass species. Inoculated plots were evaluated visually and using digital image analysis (DIA) for disease development over time and for number and area of infection centers at two rating dates each year. Statistical differences among the seashore paspalum genotypes and inoculation/isolate treatments were detected for area under the disease progress curve (AUDPC) values, number of infection centers, and infection center area. A significant interaction between seashore paspalum genotype and S. homoeocarpa isolate effects was not observed, indicating that host plant resistance genes are likely not isolate specific. Using this information, breeders should be able to use one highly virulent S. homoeocarpa isolate to screen for host plant resistance in seashore paspalum.

Dollar Spot in Four Bentgrass Cultivars as Affected by Acibenzolar-S-Methyl and Organic Fertilizers

Golf course superintendents are interested in identifying ways to reduce fungicide inputs on creeping bentgrass (Agrostis palustris Huds.) putting greens. Our objectives were to evaluate the influence of the plant defense activator acibenzolar-S-methyl (ASM, Actigard, Syngenta Corp., Basel, Switzerland) and organic fertilizers on dollar spot (Sclerotinia homoeocarpa F.T. Bennett) and brown patch (Rhizoctonia solani Kuhn) in four bentgrass cultivars. Crenshaw had over 10 times the area under the disease progress curve (AUDPC) value for dollar spot compared to the cultivar L-93 in 2000. Acibenzolar-S-methyl applied at 35 g a.i. per ha every 14 days between mid-May and mid-September for two consecutive growing seasons reduced the number of S. homoeocarpa infection centers by 15% in Crenshaw, 24% in Penncross, and 29% in Providence, but had no effect on the number of infection centers in L-93. Only Sustane (Crenshaw in 2000) and Milorganite (Crenshaw and Providence in 2001) suppressed dollar spot compared to urea. The following fertilizers resulted in an increase in S. homoeocarpa infection centers: Nature Safe 8-1.3-4.2 in Crenshaw in both years, and in Providence in 2000; Nature Safe 10-0.9-6.6 in Crenshaw in both years; and Sustane with Iron in Crenshaw in 2001. All other organic fertilizer × cultivar combinations resulted in dollar spot levels equivalent to those observed in ureatreated turf. Brown patch levels were not affected by cultivar, ASM, or organic fertilizers. The defense activator ASM deserves further evaluation as a tool to reduce dollar spot-targeted fungicide inputs in creeping bentgrass. Accepted for publication 5 June 2003. Published 26 June 2003.

Dollar Spot Resistance among Blends of Creeping Bentgrass Cultivars

Disease is a major problem in creeping bentgrass (Agrostis stolonifera L.) on golf course putting greens. Genetic composition and uniformity of host populations can affect disease development. This study was conducted to determine the effects of blending creeping bentgrass cultivars on dollar spot (caused by Sclerotinia homoeocarpa F. T. Bennett) severity. ‘Penn A‐4’, ‘Crenshaw’, ‘L‐93’, ‘Mariner’, and ‘Penncross’ were planted in monostands or as two‐way or three‐way blends in a completely random design with three replications on a sand‐based putting green in the field at College Station, TX. Disease severity was determined by the number of infection centers and by total blighted area. Crenshaw was most susceptible, L‐93 most resistant, and all other cultivars were moderately resistant to dollar spot in monostands. Cultivar susceptibility in monostands to S. homoeocarpa was representative of each cultivars contribution to dollar spot severity in blends. In blends, L‐93 suppressed, whereas Crenshaw increased dollar spot. Penncross, Penn A‐4, and Mariner did not affect dollar spot severity across all blend combinations. Number of infection centers and infected area pooled across all monostands were similar when compared to all two‐ and three‐way blends indicating that dollar spot activity in blends was a compromise between resistance levels of individual cultivars. Two‐way blends of Penn A‐4, L‐93, Mariner, or Penncross with Crenshaw, however, resulted in 46 to 67% fewer infection centers and 71 to 98% less blighted area, when compared to the Crenshaw monostand. These data indicated that blending a resistant cultivar with a susceptible cultivar reduced dollar spot severity in the blended population.

Equine gammaherpesvirus 2 (EHV2) is latent in B lymphocytes.

Peripheral blood leukocytes were collected from 5 Thoroughbred horses and examined for the presence of EHV2 in sub-populations of mononuclear cells. Peripheral blood mononuclear cells were separated on Percoll gradients and then enriched for plastic adherent cells (predominantly monocytes), surface immunoglobulin positive (sIg+) B lymphocytes and T lymphocytes, using panning techniques. The purity of each cell population was assessed by fluorescence activated cell scanning. In an infectious centre assay, each cell population was inoculated onto equine foetal kidney monolayer cell cultures which are fully permissive for the replication of EHV2. Only enrichment for sIg+ B lymphocytes resulted in a marked increase in the number of infectious centres, indicating that EHV2 is present in B lymphocytes. Freeze-thawing of sIg+ B lymphocytes, prior to inoculation onto EFK monolayer cell cultures, resulted in the complete abrogation of infectious centre formation, confirming that EHV2 is latent in B lymphocytes i.e., infectious free virus was not present in the cells. The number of EHV2 infected B lymphocytes varied considerably between horses from 4 to 780 per 10(6) cells. Evidence was also obtained that direct cell to cell contact between the epithelial cells and sIg+ B lymphocytes was necessary for the production of infectious centres. The data indicate that EHV2, like other members of the Gammaherpesvirinae, is latent within B lymphocytes.

Mechanism of Differences in Pathogenicity between Two Variants of a Laboratory Strain of Herpes Simplex Virus Type 1

The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV-1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the -GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated -GCr variant. When the attenuated variant was preinoculated i.p. 24 hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV-1 was shown to be age-dependent.

Establishment of herpes simplex virus latency in vitro with cycloheximide.

Human embryonic lung cells were infected with herpes simplex virus (HSV), treated with 10 micrograms/ml or more of cycloheximide for 24 h, incubated at 37 degrees C, and then shifted to 40.5 degrees C for various periods of time (0 to 40 days) without cycloheximide treatment. No infectious virus was detected after freezing and thawing of the cultures; however, infectious virus was recovered after temperature shift-down to 37 degrees C or superinfection with human cytomegalovirus (HCMV). The time course for formation of infectious centres after temperature shift-down was examined with and without HCMV superinfection during incubation at 40.5 degrees C. Two patterns of latently infected cells were identified: one pattern showed spontaneous reactivation of virus after temperature shift-down, and the second showed reactivation of HSV after superinfection with HCMV. The first pattern showed a rapid decrease in the number of infectious centres with time, whereas the second maintained a steady reactivation rate up to 40 days at 40.5 degrees C. The same tendency was observed for infectious centre formation at 37 degrees C with and without HCMV superinfection in the HSV latency system established with (E)-5-(2-bromovinyl)-2'-deoxyuridine and interferon treatment.

Prophage induction by ultraviolet light in Acinetobacter calcoaceticus.

UV-induction of prophage P78 of Acinetobacter calcoaceticus increased with the UV-dose given to the lysogenic strain from the spontaneous induction frequency of about 0.8% to a maximal frequency of 10%. This 10- to 20-fold increase of induction frequency, as measured by the number of infective centres, was accompanied by a 1000-fold increase in the yield of free phage. This effect was probably due to an increase in burst size under the conditions of lysogenic induction. Unusually, the lysogen was more resistant to UV-irradiation than the corresponding non-lysogenic strain.

Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug.

WIN 51711, a potent new antipicornavirus drug, has been shown to inhibit an early event in the replication cycle of human poliovirus type 2 and human rhinovirus type 2. WIN 51711 was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labeled virions to cells. When virion penetration of the plasma membrane was determined through loss of sensitivity to neutralizing antisera, WIN 51711 had no effect on poliovirus penetration, but inhibited rhinovirus penetration by 40%. In the presence of WIN 51711, exposure of neutral red-encapsidated virus-infected cells to light at 3 h postinfection resulted in a 3-log reduction in the number of infectious centers, indicating that WIN 51711 maintained the viral RNA in the encapsidated state after penetrating the cell membrane. The inhibition of uncoating by WIN 51711 in the neutral red assay was found to be concentration dependent, with a concentration of 0.03 micrograms/ml resulting in a 90% inhibition of uncoating. Sucrose gradient sedimentation of lysates from whole cells infected with [3H]uridine-labeled poliovirus showed that poliovirions remained intact in the presence of WIN 51711, but were uncoated in the absence of drug. WIN 51711 also prevented thermal inactivation of poliovirus infectivity, indicating a direct stabilizing effect of this compound on virion capsid conformation.

An efficient method for the introduction of viral DNA into Brevibacterium lactofermentum protoplasts.

A method for the introduction of a bacteriophage DNA into Brevibacterium lactofermentum protoplasts is described. Frequencies of 10(5) infective centres per micrograms DNA were easily achieved, the relationship between the number of infective centres and the amount of DNA being linear up to 5 micrograms DNA per assay. This method can be used to introduce foreign DNA into these bacteria.

Rhizobium Infection and Nodule Development in Soybean Are Affected by Exposure of the Cotyledons to Light

The initiation of Rhizobium infections and the development of nodules on the primary root of soybean Glycine max L. Merr cv Williams seedlings are strongly affected by exposure of the cotyledons/hypocotyls to light. Seedlings in plastic growth pouches were inoculated with R. japonicum in dim light and the position of the root tip of each seedling was marked on the face of the pouch. The pouches were covered and kept in the dark for various times before exposing the upper portions of the plants (cotyledons and hypocotyls) to light. Maximum nodulation occurred if the plants were kept in the dark until 1 day after inoculation. The exposure of plants to light 2 days before inoculation reduced the number of nodules by 50% while the number of nodules was reduced by 70% if the plants were kept in the dark until 7 days after inoculation. Anatomical studies revealed that exposure to light prior to inoculation reduced both the number of infection centers with visible infection threads and the number of infections which developed nodule meristems. Plants kept in the dark for 7 days after inoculation formed a normal number of infection threads above the root tip mark, but very few of these infections developed a nodule meristem. It appears that light stimulates soybean to produce substances which can both inhibit the formation of infection threads and enhance the development of nodules from established infection threads. The effects of light on nodulation appear to be expressed independently of the Rhizobium-induced suppression of nodule formation in younger regions of the root.

Experimental infection of inbred mice with herpes simplex virus. VI. Effect of interferon on in vitro virus replication in macrophages.

Peritoneal exudate cells (PEC) of DBA/2 mice, after 7 days of in vitro preculture and consisting of virtually 100 per cent macrophages, were able to support the replication of Herpes Simplex Virus type 1 strain WAL (HSV). Using a standard medium based on Dulbecco's Modified Eagle Medium (D-MEM), no virus replication was observed in freshly isolated PEC. However a medium based on RPMI 1640 consistently yielded higher virus titres in precultured PEC than the D-MEM medium, and also allowed virus replication in freshly isolated PEC. Macrophages derived from the spleens or the bone marrow, and precultured in the same way as PEC represented a highly pure population and were permissive for infection with HSV. Titres of about 10(6) PFU HSV were observed in PEC 48 hours after infection with 10(3) or 10(6) PFU. However, whereas a complete destruction of the cell monolayer was observed 24 hours after infection with 10(6) PFU, complete cytopathogenicity in PEC infected with 10(3) PFU required at least twice this time. In the latter situation, plaque formation was observed 24 hours after infection. PEC of different strains of mice were compared. Of these, PEC of all mice that are susceptible to HSV infection in vivo replicated HSV to the same degree as PEC of DBA/2 mice, whereas PEC of resistant C57BL/6 and C3H/HeJ mice produced 1000 fold lower titres of viral progeny. Whereas the number of infectious centres were equal in PEC of DBA/2 and C57BL/6 mice, the plaques observed after infection of confluent PEC with a low MOI were considerable smaller in cells from C57BL/6 mice. Furthermore, significantly higher titres of interferon were measured in the supernatants of HSV-infected C57BL/6 macrophages than in those of DBA/2 macrophages, and the former were made fully susceptible by the in vitro addition of an anti-interferon serum.

Induction of prophage .LAMBDA. in ultraviolet light sensitive ruv mutants of Escherichia coli K-12.

Induction of prophage λ in ultraviolet light sensitive ruv mutants of Escherichia coli K-12 was studied. When the ruv+ thy (λ) strain was starved for thymine, a maximum induction occurred after about 150min, whereas induction of the ruv thy(λ) strains reached the same level at about 90min. The number of infective centers in a ruv+tif (λ) strain reached a maximum at 60min after the temperature was raised to 42°C, whereas a maximum induction was achieved at 45min in the ruv tif (λ) strains. These results suggest that the ruv mutations affect the process of prophage induction in thy auxotrophs and in tif strains.

Isolation and characterization of host range mutants of avian sarcoma virus.

Two host range mutants of avian sarcoma virus (ASV) were isolated from a clone of the Bratislava 77 strain, subgroup C (B77-C). An HR- mutant was obtained by adaptation to duck cells and showed almost the same efficiencies of transformation (e.o.t.) and growth in cultures of chick embryo fibroblasts (CEF) and duck embryo fibroblasts (DEF). An HR+ mutant showed lower e.o.t. and slower growth on DEF than on CEF. Characterization of these mutants and wild-type B77-C showed that (i) unlike the host range mutants reported previously, these mutants and wild-type B77-C have common antigenicity, (ii) the number of infective centres is almost the same for the two mutants and the wild-type, and so the adsorption and integration of these viruses are similar, and (iii) the content of viral RNA in DEF infected with HR- is increased and decreased in the case of HR+ compared with the wild-type.

Role of macrophage oxidative metabolism in resistance to vesicular stomatitis virus infection.

The role of oxygen metabolites in mediating virucidal activity was studied in two cloned macrophage-like cell lines. The parental cell line, J774.16, upon appropriate stimulation with either phorbol myristate acetate (PMA) or aggregated immunoglobulin, is induced to oxidize glucose via the hexose monophosphate shunt and produce O2- and H2O2. A variant derived from it, clone C3C, is defective in oxidative metabolism and cannot be stimulated to produce O2- or H2O2. Significant differences in yields of vesicular stomatitis virus (VSV) between stimulated clone 16 cells and unstimulated cells could be obtained only when low multiplicities were used for infection. Under the same conditions, PMA stimulation of the variant clone C3C produced no reduction in yields. The effect of PMA on virus yields in clone 16 was short-lived and dose dependent. PMA stimulation of either cell line had no effect on the number of infectious centers, suggesting that the antiviral effect was likely to be an extracellular, rather than an intracellular, one. Using glucose oxidase plus aglucose to generate H2O2 in solution, we observed that H2O2 alone is capable of killing limited amounts of VSV. The inactivation of VSV, both by H2O2 in solution and by activated clone 16 cells, could be inhibited by catalase. We conclude that intracellular resistance to VSV is primarily mediated through nonoxidative mechanisms, since activated macrophages can kill only a limited number of infectious virus particles extracellularly by means of secreted H2O2.

Restricted replication of mouse adenovirus strain FL in mouse Balb/c cells transformed by Simian Adenovirus 7

Mouse kidney cells (Balb/c cells), in which mouse adenovirus strain FL (Ad FL) replicates, become restricted in their capacity to support Ad FL growth after transformation by simian adenovirus 7 (SA7). In transformed cells (Balb/c SA7 cells), the duration of the Ad FL cycle is significantly longer than in normal cells; some virus DNA replication and virus protein synthesis occur but the yield of infectious Ad FL particles is greatly reduced. Only a few cells produce virus as estimated by the number of infectious centers. However, no significant differences are noted in the adsorption and in the penetration of Ad FL in both cell types. Thus, the restrictive event in virus multiplication in Balb/c SA7 cells occurs probably after viral uncoating and before viral DNA synthesis.

Charakterisierung eines CaCl2-abhängigen Transfektionssystems vonEscherichia coli und T3-Phagen-DNS

Transfection by DNA isolated from bacteriophage T3 was studied using Escherichia coli 921/0 as host. The following conditions were found optimal: Competent E. coli 921/0 were obtained by harvesting the bacteria at the onset of late exponential growth (5 X 10(8) cells/ml) and treating the latter with 0.05 M CaCl2. Hereafter, the microbes were suspended in 50 mM Tris-HCl buffer (pH 7.2) and the concentration adjusted to 7 X 10(9) cells/ml. T3 DNA was added and the suspension kept at 0 degrees C for 15 min. Determination of the number of infectious centers was then carried out in the usual way. The efficiency of transfection under these conditions amounted to 10(4) p. f. u./microgram DNA. Preincubation of competent bacteria with T4 DNA at 0 degrees C before the addition of T3 DNA reduced the number of infectious centers. However, if T3- and T4 DNA were added simultaneously no decrease of the transfection efficiency occurred. Calf thymus DNA was without influence on transfection.

Mechanisms of herpes simplex virus infectivity enhanced by ultracentrifugal inoculation.

Ultracentrifugation of very dilute suspensions of herpes simplex virus directly onto monolayer cells grown in centrifuge tubes was studied. Enhanced infectivity by ultracentrifugation was similar at 4 degrees C and at 35 to 37 degrees C. The high infectivity levels of cultures centrifuged at 4 degrees C were further examined by infectious center assays. At 4 degrees C, the numbers of infectious centers in control (noncentrifuged) cultures were almost 100-fold fewer than in control cultures at 37 degrees C. However, the numbers of infectious centers in cultures ultracentrifuged at 4 degrees C were similar to those ultracentrifuged at 37 degrees C. The great difference in the numbers of infectious centers between 4 and 37 degrees C control cultures, in contrast to the similarity between 4 and 37 degrees C ultracentrifuged cultures, indicated that ultracentrifugation at 4 degrees C enhanced infectivity possibly by facilitation of herpes simplex virus penetration into monolayer cells.

Defective influenza viral ribonucleoproteins cause interference.

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.

The role of recombination in transfection of B. subtilis.

A comparative study of transfection with four different phage DNAs is being presented. Two types of transfection systems are distinguished, one with nearly linear dependence of the number of infective centers produced on the concentration of the phage DNA, the other type displaying multihit dose response. Studies of genetic recombination in transfection show that in systems of the latter type two (SPP 1) or three (SP 50) input genomes have to cooperate in a recombination event prior to replication. This obligatory process, termed primary recombination, is exclusively mediated by the host recombination system and cannot be effected by the phage recombination system.