A standardized approach to the evaluation of antivirals against DNA viruses: Orthopox-, adeno-, and herpesviruses.

The search for new compounds with a broad spectrum of antiviral activity is important and requires the evaluation of many compounds against several distinct viruses. Researchers attempting to develop new antiviral therapies for DNA virus infections currently use a variety of cell lines, assay conditions and measurement methods to determine in vitro drug efficacy, making it difficult to compare results from within the same laboratory as well as between laboratories. In this paper we describe a common assay platform designed to facilitate the parallel evaluation of antiviral activity against herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, vaccinia virus, cowpox virus, and adenovirus. The automated assays utilize monolayers of primary human foreskin fibroblast cells in 384-well plates as a common cell substrate and cytopathic effects and cytotoxicity are quantified with CellTiter-Glo. Data presented demonstrate that each of the assays is highly robust and yields data that are comparable to those from other traditional assays, such as plaque reduction assays. The assays proved to be both accurate and robust and afford an in depth assessment of antiviral activity against the diverse class of viruses with very small quantities of test compounds. In an accompanying paper, we present a standardized approach to evaluating antivirals against lymphotropic herpesviruses and polyomaviruses and together these studies revealed new activities for reference compounds. This approach has the potential to accelerate the development of broad spectrum therapies for the DNA viruses.

Validation of the Filovirus Plaque Assay for Use in Preclinical Studies.

A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing Biosafety Level 4 (BSL-4) studies. After standardization studies were completed, Good Laboratory Practices (GLP)-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of the Marburg Virus Angola (MARV) variant and Ebola Virus Kikwit (EBOV) variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to ±0.5 log10 PFU/mL. Repeatability precision, intermediate precision and reproducibility precision were sufficient to return viral titers with a coefficient of variation (%CV) of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay's precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (NHP) serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.

Standardization of the methods and reference materials used to assess virus content in varicella vaccines.

BACKGROUND: In Korea, every vaccine lot is tested by the National Center for Lot Release (NCLR) in accordance with the national lot release procedures to ensure the safety and efficacy of vaccines. These quality tests examine the virus content in varicella vaccines via plaque assays (either the agar overlay method [AOM] or plaque staining method [PSM]), according to the procedures suggested by the Korean Reference Material for the Varicella Vaccine (KRMVV) or the manufacturer's standard in-house protocol. AIM: To standardize the virus content tests, viral titers in the KRMVV were measured using the PSM at four participating laboratories in a collaborative study. With the aim of developing a standardized method using the KRMVV as a positive control, we compared the ability of the two test methods, AOM and PSM, to accurately and reproducibly determine the virus content of two commercial varicella vaccines. RESULTS: The results showed that the standardized method (PSM) was more suitable for quality control analysis of the varicella vaccine. CONCLUSION: Use of a standardized method (PSM) according to the Korean reference material will improve the reliability and objectivity of lot release testing.

Variability in dengue titer estimates from plaque reduction neutralization tests poses a challenge to epidemiological studies and vaccine development.

BACKGROUND: Accurate determination of neutralization antibody titers supports epidemiological studies of dengue virus transmission and vaccine trials. Neutralization titers measured using the plaque reduction neutralization test (PRNT) are believed to provide a key measure of immunity to dengue viruses, however, the assay's variability is poorly understood, making it difficult to interpret the significance of any assay reading. In addition there is limited standardization of the neutralization evaluation point or statistical model used to estimate titers across laboratories, with little understanding of the optimum approach. METHODOLOGY/PRINCIPAL FINDINGS: We used repeated assays on the same two pools of serum using five different viruses (2,319 assays) to characterize the variability in the technique under identical experimental conditions. We also assessed the performance of multiple statistical models to interpolate continuous values of neutralization titer from discrete measurements from serial dilutions. We found that the variance in plaque reductions for individual dilutions was 0.016, equivalent to a 95% confidence interval of 0.45-0.95 for an observed plaque reduction of 0.7. We identified PRNT75 as the optimum evaluation point with a variance of 0.025 (log10 scale), indicating a titer reading of 1∶500 had 95% confidence intervals of 1∶240-1∶1000 (2.70±0.31 on a log10 scale). The choice of statistical model was not important for the calculation of relative titers, however, cloglog regression out-performed alternatives where absolute titers are of interest. Finally, we estimated that only 0.7% of assays would falsely detect a four-fold difference in titers between acute and convalescent sera where no true difference exists. CONCLUSIONS: Estimating and reporting assay uncertainty will aid the interpretation of individual titers. Laboratories should perform a small number of repeat assays to generate their own variability estimates. These could be used to calculate confidence intervals for all reported titers and allow benchmarking of assay performance.

Detection of Mycobacterium avium subsp. paratuberculosis in bulk tank milk by combined phage-PCR assay: evidence that plaque number is a good predictor of MAP.

Conventional culture and a rapid phage-PCR method were used to detect Mycobacterium avium subsp. paratuberculosis (MAP) in bulk tank milk (BTM) samples. Only two of 225 samples (0.9%) were found to contain MAP by culture whereas 50 (22%) MAP-positive samples were identified using the phage-PCR assay, including both samples that were MAP-culture positive. Results using the phage-based method for independently tested duplicate samples indicated that the assay is very reproducible (r(2)=0.897), especially when low levels of mycobacteria are present. A relationship was established between plaque number and the presence of MAP in a sample. A cut-off value was determined allowing identification of MAP-positive samples based on plaque number alone (90% sensitivity, 99% specificity; area under the curve=0.976). These results indicate that the assay is a robust method for screening BTM, providing results within 24 h.

Optimization and validation of a plaque reduction neutralization test for the detection of neutralizing antibodies to four serotypes of dengue virus used in support of dengue vaccine development.

A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype-specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT(50) for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay. The PRNT(50) was then validated and demonstrated to be suitable to detect and measure dengue serotype-specific neutralizing antibodies in human serum samples with acceptable intra-assay and inter-assay precision, accuracy/dilutability, specificity, and with a lower limit of quantitation of 10.

Standardization of the filovirus plaque assay for use in preclinical studies.

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.

Variation in dengue virus plaque reduction neutralization testing: systematic review and pooled analysis.

BACKGROUND: The plaque reduction neutralization test (PRNT) remains the gold standard for the detection of serologic immune responses to dengue virus (DENV). While the basic concept of the PRNT remains constant, this test has evolved in multiple laboratories, introducing variation in materials and methods. Despite the importance of laboratory-to-laboratory comparability in DENV vaccine development, the effects of differing PRNT techniques on assay results, particularly the use of different dengue strains within a serotype, have not been fully characterized. METHODS: We conducted a systematic review and pooled analysis of published literature reporting individual-level PRNT titers to identify factors associated with heterogeneity in PRNT results and compared variation between strains within DENV serotypes and between articles using hierarchical models. RESULTS: The literature search and selection criteria identified 8 vaccine trials and 25 natural exposure studies reporting 4,411 titers from 605 individuals using 4 different neutralization percentages, 3 cell lines, 12 virus concentrations and 51 strains. Of 1,057 titers from primary DENV exposure, titers to the exposure serotype were consistently higher than titers to non-exposure serotypes. In contrast, titers from secondary DENV exposures (n = 628) demonstrated high titers to exposure and non-exposure serotypes. Additionally, PRNT titers from different strains within a serotype varied substantially. A pooled analysis of 1,689 titers demonstrated strain choice accounted for 8.04% (90% credible interval [CrI]: 3.05%, 15.7%) of between-titer variation after adjusting for secondary exposure, time since DENV exposure, vaccination and neutralization percentage. Differences between articles (a proxy for inter-laboratory differences) accounted for 50.7% (90% CrI: 30.8%, 71.6%) of between-titer variance. CONCLUSIONS: As promising vaccine candidates arise, the lack of standardized assays among diagnostic and research laboratories make unbiased inferences about vaccine-induced protection difficult. Clearly defined, widely accessible reference reagents, proficiency testing or algorithms to adjust for protocol differences would be a useful first step in improving dengue PRNT comparability and quality assurance.

Infection on a chip: a microscale platform for simple and sensitive cell-based virus assays.

The plaque assay has long served as the "gold standard" to measure virus infectivity and test antiviral drugs, but the assay is labor-intensive, lacks sensitivity, uses excessive reagents, and is hard to automate. Recent modification of the assay to exploit flow-enhanced virus spread with quantitative imaging has increased its sensitivity. Here we performed flow-enhanced infection assays in microscale channels, employing passive fluid pumping to inoculate cell monolayers with virus and drive infection spread. Our test of an antiviral drug (5-fluorouracil) against vesicular stomatitis virus infections of BHK cell monolayers yielded a two-fold improvement in sensitivity, relative to the standard assay based on plaque counting. The reduction in scale, simplified fluid handling, image-based quantification, and higher assay sensitivity will enable infection measurements for high-throughput drug screening, sero-conversion testing, and patient-specific diagnosis of viral infections.

Accuracy and repeatability of a micro plaque reduction neutralization test for vaccinia antibodies.

The detection of neutralizing antibodies against vaccinia virus is a valuable tool for the investigation of previous smallpox vaccination. Compulsory smallpox vaccination ended in Brazil during the early 1970s, although the vaccine was available until the late 1970s. The threat of smallpox as a biological weapon has called the attention of public health authorities to the need for an evaluation of the immune status of the population. Based on our previous experience with a micro plaque reduction neutralization test (PRNT) for the evaluation of yellow fever immunity, a similar test was developed for the detection and quantification of vaccinia neutralizing antibodies. A cross-sectional study to test the repeatability and validity of plaque reduction neutralization test (PRNT) for vaccinia antibodies was performed in 182 subjects divided into two categories: subjects above 31 years old and the other > or = 35 years old. Cases were subjects considered to have been vaccinated with vaccinia virus if they declared vaccination history or evidenced vaccination marks. The assay is carried out in 96-well plates, provides results within 30 h, is easily performed, has good sensitivity (92.7%) and specificity (90.8), excellent repeatability (ICC 0.89 (0.88; 0.92)) and is thus suitable for use in mass screening of a population's antibody levels.

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context.

We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how to measure the degree of chromatin assembly in the absence and presence of histone H1 using topological analysis and how to perform micrococcal nuclease digestion to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further, we describe the use of sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).

Inactivation of a European strain of West Nile virus in single- donor platelet concentrate using the INTERCEPT blood system.

BACKGROUND AND OBJECTIVE: In order to prevent West Nile virus (WNV) contaminations by transfusion, the French National Blood Service decided to evaluate the INTERCEPT Blood System's efficiency on a European strain. MATERIALS AND METHODS: Culture supernatant of WNV was used to infect six platelets concentrates. Viral titre was determined by plaque reduction neutralization test before and after viral inactivation using the INTERCEPT Blood System. RESULTS: In all assays, the absence of plaque forming unit was observed after viral inactivation. The log reduction observed ranged between > 5.1 logs to > 5.2 logs. CONCLUSION: INTERCEPT Blood System is a commercially viral inactivation method potentially useful in order to prevent WNV transmission by blood products in France during re-emerging outbreaks.

Efficacy of antiviral agents in feline herpetic keratitis: results of an in vitro study.

PURPOSE: To determine, by a plaque reduction assay, the in vitro efficacy of novel antiviral agents in the treatment of feline herpes virus 1 (FHV-1) keratitis in the domestic cat (Felis felis). MATERIALS AND METHODS: A standard plaque reduction assay was performed using a laboratory strain of FHV-1 and embryo-derived feline kidney cells to determine the in vitro efficacy of the antiviral drugs penciclovir (PCV), bromovinyldeoxyuridine (BVdU), and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) and to compare these with the drugs acyclovir (ACV) and trifluorothymidine (TFT). Efficacy was assessed by determining the dose of drug at which 50% plaque reduction was noted (ED(50)). RESULTS: HPMPA was found to have greatest antiviral activity (ED(50) 0.07 microg/ml). ACV was least active (ED(50) 24 microg/ml), while TFT was active with an ED(50) of 5.7 microg/ml. PCV and BVdU had intermediate activity (ED(50) 1.6 and 1.7 microg/ml, respectively). CONCLUSIONS: This study suggests that the efficacy of HPMPA, BVdU, and penciclovir in cats with herpesviral keratitis should be determined in vivo as their efficacy in vitro was substantially greater than that of acyclovir, already shown to have demonstrable but limited clinical antiviral activity.

A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates.

Twelve laboratories collaborated in formulating and testing a standardized plaque reduction assay for cytomegalovirus (CMV) cell-associated clinical isolates. Four characterized and plaque-purified CMV strains, as well as six coded clinical isolates obtained after antiviral therapy, were distributed and tested. Good agreement was obtained for four of the clinical isolates, but a broad distribution of results was obtained for two isolates. Analysis of these results indicates the problems associated with clinical isolates, including the large genetic variability and the highly cell-associated phenotype. This collaborative effort, by addressing these problems, represents a significant step toward the development of a standardized assay.

PG-4 cell plaque assay for xenotropic murine leukemia virus.

Xenotropic murine leukemia virus (X-MuLV) is often used in retrovirus elimination studies involving rodent cells. Currently, X-MuLV is measured using a focus-forming assay on mink (MiCl1 S+L-) or cat (PG-4 S+L-) cell lines. An easier and quicker PG-4 cell plaque assay, which retains the statistical reproducibility of the focus-forming assay, was developed and evaluated in this study. The PG-4 plaque assay is more sensitive than the MiCl1 focus assay for titering X-MuLV. The best results were achieved by passaging PG-4 cells at a seeding density of 4x10(6) cells/T185 flask twice a week, inoculating 3x10(5) cells/well on six-well plates and performing the assay at 35 degrees C. The overall variability of the assay was 0.30 log10 titer unit. A linear response to dilution was observed for wells containing 5-70 plaques. The limit of quantitation was 10 PFU/ml. Using six wells per dilution, the 95% confidence limit of the grand mean titer was within +/-0.5 log10.

RT-PCR and cell culture infectivity assay to detect enteroviruses during drinking water treatment processes.

In this study, 62 water samples were collected from two water treatment plants (WTPs) in Suez Canal cities (Port Said and Ismaillia) and one plant in Cairo (Giza WTP) in addition to the beginning of the two Nile river branches (Rosetta and Damietta). Viruses were concentrated by adsorption-elution ethod sing 142 mm-diameter nitrocellulose membrane of 0.45 microm pore size and eluted with 3% beef extract at pH 9.5. The concentrated samples were inoculated for 3 successive passages in three cell culture types (Vero, BGM and RD). Enterovirus RNAs in CPE-induced samples were extracted by guanidinium thiocyanate/ phenol/chloroform and heat shock methods and detected by RT-PCR and neutralization test. The results showed that eight samples [14.5% (8/62)] contained enteroviruses most of them were polioviruses [87.5% (7/8)] and coxsackievirus type B2 [12.5% (1/8)]. The three cell cultures were of the same sensitivity to detect the isolated viruses. Also, RT-PCR followed by neutralization assay facilitates and accelerate the results. The guanidinium thiocyanate extraction method was more sensitive than heat shock method. The results turned our attention to review our technology of water treatment and disinfection step in addition to the selection of suitable intake for the drinking water treatment plants.

[The standardization of the plaque reduction technic for differentiating a dengue infection from a yellow fever infection]. / Normalización de la técnica de reducción de placas para diferenciar una infección por dengue de una infección por fiebre amarilla.

The plaque reduction neutralization assay was standardized to differentiate an infection caused by dengue from another infection produced by yellow fever. Serum samples from Cuban donors were used to this end. Information on previous vaccination against yellow fever was available. Samples from Costa Rican patients with a clinical picture of dengue and with no antecedents of vaccination against yellow fever were also utilized. The optimal plaque staining day was the fifth day and the smallest serum dilution capable of differentiating an infection resulting from dengue from another infection caused by yellow fever was of 1/5. According to the high specificity of the standardized technique, risk factor studies of dengue hemorrhagic fever could be made among individuals vaccinated against yellow fever, which is a present and important topic.

An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages.

This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis. Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage-host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.

[Standardization of the plaque method for viruses isolated in patients with epidemic neuropathy]. / Normalización del método de placas para los virus aislados en pacientes con neuropatía epidémica.

This paper reports on the necessary conditions for plaque development in mild cutopathogenic effect-producing agents which were isolated from samples of the cerebrospinal fluid of patients presenting with epidemic neuropathy.

Comparative study on the plaque forming cell response by two different methods of plaque assay.

The Cunningham and Sezenberg Technique (using mice sensitized lymphoid cells to SRBCs) was tested together with Trump's gel technique (using non sensitized mice lymphoid cell mixed with SRBCs and incubated in Marbrook culture chamber for sensitization). The study showed that the number of PFCs by the Cunningham slid was greater than that observed by the gel technique of Trump at the same lymphocyte concentration. Also it was observed that the Trump's method using the Marbrook tissue culture chamber was a time consuming technique than that obtained by the Cunningham technique.