Number Of Plaque-forming Units Research Articles

Rapid and stain-free quantification of viral plaque via lens-free holography and deep learning

A plaque assay—the gold-standard method for measuring the concentration of replication-competent lytic virions—requires staining and usually more than 48 h of runtime. Here we show that lens-free holographic imaging and deep learning can be combined to expedite and automate the assay. The compact imaging device captures phase information label-free at a rate of approximately 0.32 gigapixels per hour per well, covers an area of about 30 × 30 mm2 and a 10-fold larger dynamic range of virus concentration than standard assays, and quantifies the infected area and the number of plaque-forming units. For the vesicular stomatitis virus, the automated plaque assay detected the first cell-lysing events caused by viral replication as early as 5 h after incubation, and in less than 20 h it detected plaque-forming units at rates higher than 90% at 100% specificity. Furthermore, it reduced the incubation time of the herpes simplex virus type 1 by about 48 h and that of the encephalomyocarditis virus by about 20 h. The stain-free assay should be amenable for use in virology research, vaccine development and clinical diagnosis.

Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.

Modeling Asthma in Mice Using Rhinovirus Infection.

Rhinovirus (RV) infection is linked to early life wheezing and exacerbation of adult asthma. RV infection can be modeled in adult and neonatal mice. This chapter outlines methods for the production of standardized human rhinovirus A1B and mouse infection. The chapter also describes methods to couple infections with allergen (ovalbumin and house dust mite) administrations. The production of the virus involves its amplification, purification, and concentration. In order to standardize the concentrated RV stock, a plaque assay on HeLa cells is outlined as a method of calibrating infectivity. Once the number of plaque-forming units is determined, the standardized virus is used for mouse infection.

Antiviral effect of honey extract Camelyn against SARS-CoV-2

This study aimed to evaluate the potential antiviral effects of honey extract “Camelyn” against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). The baby hamster kidney cell line 21 (BHK‐21), bone marrow-derived hematopoietic stem cells (HSCs), and splenic cells were used for Camelyn cytotoxicity assay. After the isolation procedures, cell viability was assessed by trypan blue dye exclusion under a microscope using a hemocytometer. The in vitro cell growth rate was carried out using the cell counting Kit 8 (CCK-8) assay. The cells were seeded in growth media with various Camelyn concentrations (35 μg, 50 μg, 70 μg, 100 μg, 150 μg, and 200 μg). The absorbance at 450 nm was determined by the multiplate reader. The antiviral effect was assessed by plaque reduction assay for the determination of drug susceptibility against SARS-CoV-2. Serial dilution of the selected compounds was pre-incubated with 40 to 100 plaque-forming units (PFUs) of SARS-CoV-2. The pre-incubated mix of Camelyn and SARS-CoV-2 was then added to the confluent Vero E6 cells after incubation cells were fixed and stained and the number of PFUs was counted under an inverted microscope and plotted against the logarithm of antiviral concentrations. Our study showed that Camelyn is not cytotoxic, has a stimulatory effect on cell proliferation, and has an inhibitory effect against SARS-CoV-2 with EC50 (half-maximal effective concentration) from 85.7 μg/mL to 192.4 μg/mL depending on product concentration and viral plaque per cell. © 2021 Global Research Online. All rights reserved.

Environmental persistence of equid herpesvirus type-1.

Equid herpesvirus type 1 (EHV-1) is ubiquitous in equine populations causing respiratory disease, and complications including late-term abortion and neurological disease. Eradication of EHV-1 from housing environments that typically contain unsealed wood and porous bedding materials can be challenging. However, consideration should be given to take advantage of the viral envelope's susceptibility to environmental conditions. To determine environmental persistence of EHV-1 on materials and in environmental conditions commonly found in equine facilities. We hypothesised that environmental conditions and materials would limit environmental persistence of EHV-1 in horse housing environments. Experimental study. Standard inoculum of EHV-1 strain OH03 was applied to leather, polyester-cotton fabric, two bedding materials (pinewood shavings and wheat straw) and polystyrene (plastic), and placed under three different environmental conditions (4°C, indoors and outdoors). Virus titration and quantitative PCR (qPCR) were performed at six time points between 0 and 48hours and the number of plaque-forming units (PFUs) was determined. Viable EHV-1 was recovered up to 48hours from all material-environmental condition combinations, with persistence decreasing over time. In general, outdoor environment had the greatest impact, irrespective of material tested, followed by indoor environment and 4°C. On average, wood shavings had the greatest impact on persistence, followed by leather, straw, fabric and polystyrene. The inoculum used in this study was not in a milieu consistent with nasal secretions. As such, virus particles may have been more sensitive to the materials and/or environmental conditions evaluated. Environmental factors had variable effects on environmental persistence. Although there were significant reductions in PFUs within the first 3hours, irrespective of environment-material evaluated, viable virus was still recovered at 48hours likely representing a transmission risk. Barrier precautions should be used to prevent spread of EHV-1 from unrecognised environmental reservoirs.

Determination of Optimum Survivability Factors of Highly Pathogenic Vibrio cholerae 01 Serogroup-Specific Bacteriophage JSF4ϕ

Cholera is severe watery diarrhea caused by pathogenic V. cholerae 01 or 0139 serogroups. In each year, 2.9 million people are affected by cholera worldwide and 95000 deaths occur from the disease annually. In Bangladesh, around 100000 people are affected by this disease and approximately 4500 deaths occur each year. In this study, a novel V. cholerae 01 serogroup-specific bacteriophage JSF4ϕ was used. This phage was able to lyse both the clinical and environmental pathogenic V. cholerae 01 serogroup strains and one of our previous studies demonstrated that the seasonal outbreaks of cholera caused by V. cholerae 01 serogroup strains in Bangladesh are mostly regulated by this bacteriophage. In this current study, we determined the optimum survivability factors of JSF4ϕ bacteriophages. This study showed that the temperature 2500C, pH 7 and normal saline are the optimal survivability factors for JSF4ϕ bacteriophages because, at these conditions, we have got the maximum number of plaque-forming units (PFU/mL) of these bacteriophages. This study also showed that the JSF4ϕ bacteriophages can survive at a wide range of temperature, pH and salinity. So, the study presented here may have an impact on the controlling of cholera epidemics caused by environmental and clinical pathogenic V. cholerae 01 serogroup strains if we can use JSF4ϕ bacteriophages as a biocontrol agent. This study may also have profound implications for future studies of JSF4ϕ bacteriophages as a good food additive or in phage therapy for its efficient lysing capacity against the pathogenic V. cholerae 01 serogroup strains.

Evaluation of Factors That Influence Dose Variability of Marek's Disease Vaccines.

Marek's disease (MD) vaccines are cell-associated and require special handling and care during administration. Vaccine dose is evaluated by plaque assay and is indicated as the number of plaque-forming units (PFUs) per dose. The objectives of this study were to evaluate the dose variability within each vial of MD vaccines and to assess those factors (from both manufacturing and handling and administration of the vaccine) that could affect vaccine dose variability. Three experiments were conducted. Experiment 1 was to evaluate dose variability in 36 MD vaccine vials and the effect of manufacturing factors on dose variability. Vaccines were titrated 10 times. Dose variability was measured as the coefficient of variability (CV) calculated as standard deviation divided by average PFU and multiplied by 100. Our results showed that all evaluated vaccines had levels of CV ranging from 10% to 34%. Variability existed regardless of manufacturer, vaccine serotype, and batch. Experiment 2 was conducted to evaluate the effect of infectivity rate (IR) on CV. IR was artificially reduced by adding noninfected chicken embryo fibroblast to the reconstituted vaccine before titration. Our results showed that decreased IR results in higher CV. Experiment 3 was to evaluate the handling and administration factors (time and mixing during administration) on CV. Our results showed that CV tends to increase with time and that this effect is more remarkable if vaccines were not mixed. Our study emphasizes the relevance of proper handling of MD vaccines and shows that dose variability can jeopardize the uniformity of vaccination in a flock and therefore the success of vaccination.

Combined Application of Essential Oil Compounds and Bacteriophage to Inhibit Growth of Staphylococcus aureus In Vitro.

Staphylococcus aureus is considered an important human pathogen. This study aimed to investigate the combination of essential oil compounds (EOCs) and bacteriophage as alternative antimicrobials to control S. aureus in vitro. Here, four EOCs (alpha-pinene, 3-carene, (+)-limonene, (1S)-(-)-β-pinene) were evaluated by disc diffusion assay (DDA) and growth inhibition assay (GIA) to determine inhibitory effects against five strains of S. aureus. Phage adsorption assays were performed with phage K up to 120 h at 6, 13, and 37 °C to determine lytic activity. Combinations of phage K and EOCs against S. aureus were also evaluated at 37 °C. Alpha-pinene exhibited significantly greater inhibition towards S. aureus strains when compared to other EOCs tested by the DDA. GIAs indicate that all S. aureus strains exhibited significantly reduced growth (P < 0.006) over a 48-h period when exposed to EOCs. Phage adsorption assays indicate that phage K has high lytic activity at 37 °C with at least a 1.5-log increase in the number of plaque-forming units (PFU) over 6 h when compared to 6 and 13 °C. S. aureus strains showed significantly reduced growth (P < 0.05) when treated with combined phage K and EOCs. Results from the combined effect of EOC and phage indicate that phage alone inhibits S. aureus in vitro at 37 °C as effectively as EOCs alone or in combination with phage with variability between strains. The results from DDA, GIA, and phage adsorption assays indicate that select EOCs and phage K can be used as antimicrobials against S. aureus in vitro with potential application in situ.

Features of membrane receptors in bacterial multiplication process and necessary conditions for phage infection of bacteria.

According to the obtained experimental results, the thermal shock (from 37 to 53 °C) not only stops the multiplication process of Escherichia coli bacteria, but also causes bacterial titer to decrease gradually. After this period lasting up to 1 hour, the bacterial cells continue to grow. A similar type of response was observed when bacteria were subjected to acid shock. Increasing acidity of media leads to decrease of bacterial growth process, and finally, their titer curve sharply falls over time. Also, interesting results were obtained about necessary conditions for infecting the bacteria by phages. Particularly, DNA injection from phages into bacterial cells requires most of corresponding bacterial membrane receptors to be occupied by phages. We suppose that this occurs due to autocrine phenomenon when the signaling molecules block the DNA ejection from phage particles. This effect lasts until a certain number of phage particles are attached to the membrane. After that, DNA injection from phage head into the cytoplasm takes place and the process of bacterial infection begins. The real number of phages in a stock is by several orders higher than the number of plaque-forming units in a given stock, which is determined by a classical double-layer agar method.

Influence of the Addition of Antibiotics on Survival of Herpesvirus of Turkeys

To determine the influence of the antibiotics ceftiofur sodium from two different laboratories (A and B) and gentamycin sulfate on a Marek's disease commercial vaccine herpesvirus of turkey (HVT), samples were assayed by titration in chicken embryo fibroblasts (CEF). Viruses were tested in vitro to establish the average number of plaque-forming units before and after different periods of incubation with the addition of the antibiotic. These tests showed no effect of gentamycin or ceftiofur A or B on HVT titers when treatments were for 1 hr or less. However, ceftiofur B decreased the titer at 2 hr. The in vivo effects of the antibiotics were determined by vaccinating 15 one-day-old chickens with HVT plus gentamycin or ceftiofur A or B. Birds were considered viremic at 1 wk postvaccination when one or more plaques were detected in CEF 5 days after inoculation of peripheral blood lymphocytes. Viremia levels were similar between 1 and 16 wk after vaccination with HVT with ceftiofur A or B. The pH values (7.5) were the same in vaccines with and without antibiotics.

QUANTITATIVE ANALYSIS OF DENGUE-2 VIRUS RNA DURING THE EXTRINSIC INCUBATION PERIOD IN INDIVIDUAL AEDES AEGYPTI

Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2-3 log(10). Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.

A hammerhead ribozyme inhibits the proliferation of an RNA coliphage SP in Escherichia coli

Ribozymes are potentially powerful tools for the suppression of intracellular gene expression. However, the few reports that exist of their activities in bacteria have described mixed success. Chuat and Galibert (Chuat, J.-C., and Galibert, F. (1989) Biochem. Biophys. Res. Commun. 162, 1025-1029) failed to detect any trans-activities of hammerhead ribozymes in Escherichia coli, while Sioud and Drlica (Sioud, M., and Drlica, K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7303-7307) reported complete inhibition of expression of the gene for a nonbacterial protein, HIV-1 integrase, by trans-acting hammerhead ribozymes in E. coli. It is of interest to determine whether ribozymes can really be used in natural bacterial systems (Altman, S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10898-10900). We now report that a ribozyme designed to cleave the A2 gene of RNA coliphage SP, when transcribed from a plasmid in E. coli caused failure of the proliferation of progeny phage. Inactive ribozymes with altered catalytic sequences did not affect phage growth. These results indicate that it is mainly the catalytic activity of the ribozyme and not its function as an antisense molecule that is responsible for suppressing the proliferation of the RNA phage. Moreover, an analysis based on numbers of plaque-forming units and the function of the A2 protein indicated that antisense RNA may successfully compete with ribosomes in targeting mRNA while ribozymes in this study may not compete with ribosomes in naturally occurring bacterial transcription/translation-coupled systems.

Structural and viral association comparisons of bovine zonae pellucidae from follicular oocytes, Day-7 embryos and Day-7 degenerated ova

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined. Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P<0.05) or with Day-7 ova (mean PFU/ovum = 31.9; P<0.01). The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.

The Occurrence of Male-Specific and Somatic Bacteriophages in Polluted South African Waters

The occurrence of somatic (F') and male-specific (F') coliphages and Salmonella phages in a variety of environmental water samples was studied using different bacterial hosts. The number of plaque-forming units (pfu) of the different bacteriophages were compared and their resistance pattern to a biological treatment (humus tank) and chlorination was evaluated. The presence of the bacteriophages in shellfish was also studied. The morphology of isolate bacteriophages was examined as well as the visibility of the different plaques formed. Coliphages were found to produce larger and clearer plaques than all other bacteriophages studied. In most of the environmental water samples coliphages outnumbered all other bacteriophages, with the exception of dam water in which higher levels of F' Salmonella phages were detected. The majority of the F' Salmonella phages were shown to be RNA bacteriophages.

Evidence that T-lymphocytes are part of the blood-brain barrier to virus dissemination

The dissemination of a neurovirulent strain of influenza A/WSN (HONI) virus from infected lungs to brains of thymus-deficient nude and immunocompetant furred mice inoculated intranasally was compared. Nude mice were more susceptible to lethal disease following intranasal (i.n.) inoculation with A/WSN virus than furred mice based on the number of plaque-forming units (PFU) of virus required to constitute a median lethal dose (LD 50). In normal mice, virus was cleared from the lungs of survivors and dissemination of virus from lung to brain was detected only rarely. NUde mice, in contrast, had frequent and early deaths with significant brain virus titers and histologic evidence of encephalitis. Adoptive immunization of recipient nude mice with suspensions of cytotoxic T-lymphocytes secondarily stimulated in vitro 24 h after i.n. challenge, reduce both brain virus titers and mortality in these animals. These data indicated that T-lymphocytes were a significant factor in preventing dissemination of neurotropic virus from lungs to the brains of infected mice.

Effect of bovine embryo quality on survival after rapid freezing and thawing

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined.Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P<0.05) or with Day-7 ova (mean PFU/ovum = 31.9; P<0.01).The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.

Dose-Dependent Inhibition of Virus Rescue from Lymphocytes Latently Infected with Turkey Herpesvirus or Marek's Disease Virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.

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The number of plaque-forming units (PFU) from lymphocytes latently infected with oncogenic (GA-5) or nononcogenic (SB-1) Marek's disease virus (MDV) was decreased after complement lysis with antisera against thymus-derived lymphocytes or bursa-derived lymphocytes; an additive effect with dual treatment was observed. Complement lysis with antisera against Marek's disease tumor-associated surface antigen (putative tumor antigen) had little effect, which suggests an absence of the antigen on latently infected cells. None of the serum complement treatments affected the number of PFU from lymphocytes infected with turkey herpesvirus (HVT). No viral antigens were detected in spleen lymphocytes when removed from infected birds. After 42-72 hours of incubation, many cultured spleen cells from MDV-infected birds (JM-10, GA-5, and SB-1 isolates) contained viral antigen, whereas very few if any cultured cells from HVT-infected birds were positive. These data suggest that the nature of the latent infections with MDV and HVT may differ significantly.

The ecology of viruses attacking Bacillus stearothermophilus in soil

The ecology of bacteriophages attacking the thermophilic soil organism Bacillus stearothermophilus has been investigated. Phage was found in every soil tested including a sample from the geothermal area of Taupo. Since the numbers of plaque-forming units (pfu) in most soils were often low a technique for enriching soil with substrate for the host cells and factors promoting phage proliferation was developed; this enabled statistically significant numbers of pfu to be scored under a variety of test conditions. Phage development in soil was greatest at 45°C while proliferation at 55°C was usually much less pronounced. These results indicate that in soil these bacteria and their associated phages grow optimally at mesophilic rather than thermophilic temperatures. It is proposed that in the field the bulk of the metabolizing population of B. stearothermophilus is mesophilic in its biochemistry and that the ‘thermophilic’ strains of this organism held by many laboratories represent selection by the isolation procedure of a minority of atypical cells.

Infectivity of herpes simplex virus particles with and without envelopes.

Herpes simplex virions produced by infected human carcinoma No. 2 (HEp-2) cells appear in two general forms, unenveloped (naked) and enveloped. These two forms were separated by zone centrifugation at 18 000 g for 55 min through a 10 to 30% (w/v) sucrose density gradient. The three bands containing virus particles were found by electron microscopy to contain empty naked particles, full naked particles, and enveloped particles. The number of virus particles and the number of plaque-forming units (p.f.u.) were determined for each of the three types of particles. In the band containing enveloped virus particles the infectivity was 1 p.f.u./120 particles; the filled naked particle band yielded 1 p.f.u./60 000 particles; and the empty naked particle band yielded 1 p.f.u./960 000 particles. All of the infectivity of the starting material was recovered from the gradient after centrifugation. It appears that the plaque-forming efficiency of naked virus particles is extremely low, but is not negligible. Particle counts of enveloped full capsids indicate that empty and filled naked particles are enveloped indiscriminately.The infectivity of naked HSV particles as well as the number of naked particles that retain a core is increased when virus is harvested in pH 8.3 medium containing 0.01 M Na citrate, conditions that are unfavorable for DNase activity. The infectivity and morphology of enveloped particles are not affected by this harvesting method.