Quantal Assay Research Articles

The influence of incubation time on adenovirus quantitation in A549 cells by most probable number

Cell culture based assays used to detect waterborne viruses typically call for incubating the sample for at least two weeks in order to ensure that all the culturable virus present is detected. Historically, this estimate was based, at least in part, on the length of time used for detecting poliovirus. In this study, we have examined A549 cells infected with human adenovirus type 2, and have found that a three week incubation of virus infected cells results in a higher number of detected viruses by quantal assay than what is seen after two weeks of incubation, with an average 955% increase in Most Probable Number (MPN) from 2 weeks to 3 weeks. This increase suggests that the extended incubation time is essential for accurately estimating viral titer, particularly for slow-growing viruses, UV treated samples, or samples with low titers of virus. In addition, we found that for some UV-treated samples, there was no detectable MPN at 2 weeks, but after 3 weeks, MPN values were obtained. For UV-treated samples, the average increase in MPN from 2 weeks to 3 weeks was 1401%, while untreated samples averaged a change in MPN of 674%, leading us to believe that the UV-damaged viral DNA may be able to be repaired such that viral replication then occurs.

Prevalence of Influenza A(H1N1)pdm09 Virus Resistant to Oseltamivir in Shiraz, Iran, During 2012 - 2013.

Background:Oseltamivir has been used as a drug of choice for the prophylaxis and treatment of human influenza A(H1N1)pdm09 infection across the world. However, the most frequently identified oseltamivir resistant virus, influenza A(H1N1)pdm09, exhibit the H275Y substitution in NA gene.Objectives:This study aimed to determine the prevalence and phylogenetic relationships of oseltamivir resistance in influenza A(H1N1)pdm09 viruses isolated in Shiraz, Iran.Patients and Methods:Throat swab samples were collected from 200 patients with influenza-like disease from December 2012 until February 2013. A total of 77 influenza A(H1N1)pdm09 positive strains were identified by real-time polymerase chain reaction (PCR). Oseltamivir resistance was detected using quantal assay and nested-PCR method. The NA gene sequencing was conducted to detect oseltamivir-resistant mutants and establish the phylogeny of the prevalent influenza variants.Results:Our results revealed that A(H1N1)pdm09 viruses present in these samples were susceptible to oseltamivir, and contained 5 site specific mutations (V13G, V106I, V241I, N248D, and N369K) in NA gene. These mutations correlated with increasing expression and enzymatic activity of NA protein in the influenza A(H1N1)pdm09 viruses, which were closely related to a main influenza A(H1N1)pdm09 cluster isolated around the world.Conclusions:A(H1N1)pdm09 viruses, identified in this study in Shiraz, Iran, contained 5 site specific mutations and were susceptible to oseltamivir.

Effects of hypo- and hyperthyroid states on herpes simplex virus infectivity in the rat

Objective: Available data from in vitro studies show that thyroid hormones (THs) regulate herpes simplex virus (HSV) gene expression and may modulate latency/reactivation of the virus. Whether infectivity of the virus is also affected by THs is not known. Using animal models (in vivo study) and Vero cell culture (in vitro study), we examined the effects of alterations in THs level on HSV-1 infectivity. Methods: Rats were rendered hypo- and hyperthyroid by daily addition of methimazole and l-thyroxine into their drinking water, respectively. Euthyroid animals served as control. All animals were given a single dose of HSV-1 (107TCID50, ip) and sacrificed 3 d later. The spleen of the animals was then removed and viral particles were recovered from the tissue extract through aseptic procedures. Serial dilution of the extracts was prepared and added to Vero cell culture. For the in vitro study, the cultures were pretreated with l-thyroxine and the viral particles were then added. Virus titration was determined by Reed–Muench quantal assay. Results: The viral load of spleen in hyperthyroid rats was significantly lower (1000-fold) than that of the euthyroid rats. Similarly, in vitro presence of supraphysiologic levels of l-thyroxine in the culture media of Vero cells decreased virus infectivity. Interestingly, hypothyroid animals showed a significant increase (10-fold) in spleen viral load as compared to that of their euthyroid counterparts. Conclusions: These data clearly show that the HSV-1 infectivity is affected by THs, and suggest that THs or their analogs may have a potential application in prevention and/or treatment of viral infections.

A note on the suitability of the Indian frog (Rana tigrina) for the biological assay of tincture of digitalis.

Abstract The relationship between the log of the dose of tincture of digitalis and the effect, in terms of probits of the mortalities (per cent) among the Indian frog, R. tigrina, has been found to be linear. Statistical analysis of the data collected from four experiments performed from July to November has not shown any non-linear relationship. The results obtained by applying the B.P. 2 and 2 quantal response assay on the frog method have been found to be in fair agreement with those obtained by using guinea pigs. The fiducial limits of error of potency of the samples at (P = 0.95) have been found to lie within the limits specified in the B.P. 1953. By using R. tigrina, potencies of the standard, substandard and abnormal samples of tincture of digitalis have been satisfactorily determined. The advantage of the frog method is the speed and simplicity of the technique: the disadvantage is the restricted availability of the suitable size of the frog during the five months July to November.

Use of the mouse jumping test for estimating antagonistic potencies of morphine antagonists.

The potencies of 19 reference morphine antagonists have been compared in a modified version of the mouse jumping test. Mice were each implanted subcutaneously with one 75 mg pellet of morphine. Antagonist challenge took place 72 h later and the incidence of repetitive vertical-jumping was monitored over 1 h. A high Pearson correlation coefficient (r = 0.997) was found between quantitative assays based on the total number of jumps per mouse and quantal assays based on mice jumping at least 6 times. A comparison of relative potencies obtained with the mouse test and with non-withdrawn morphine-dependent monkeys gave a Spearman rank order coefficient of 0.91 while a similar comparison with values obtained with the guinea-pig isolated ileum preparation also gave a high correlation coefficient (r= 0.92). Whereas it is difficult to assess the antagonistic component of buprenorphine and cyclorphan with the ileum preparation, both compounds can be satisfactorily assayed in the mouse jumping test. The reported antagonistic properties of ketocyclazocine and profadol could not be confirmed in the mouse model.

Generalized Confidence Intervals for Ratios of Regression Coefficients with Applications to Bioassays

The problem of constructing a confidence interval for the ratio of two regression coefficients is addressed in the context of multiple regression. The concept of a Generalized Confidence Interval is used, and the resulting confidence interval is shown to perform well in terms of coverage probability. The proposed methodology always results in an interval, unlike the confidence region generated from Fieller's theorem. The procedure can easily be implemented for parallel-line assays, slope-ratio assays, and quantal assays under a probit model. Furthermore, this approach can also be extended to compute confidence intervals based on data from multiple bioassays. The results are illustrated using several examples.

Development of a Cell Culture Method for Quantal Assay of Strain I-2 of Newcastle Disease Virus

Repeated titrations of strains of Newcastle disease virus (NDV) are more conveniently undertaken in cell cultures rather than in embryonated eggs. This is relatively easy with mesogenic and velogenic strains that are cytopathic to various cell lines, but is difficult with avirulent Australian isolates that are poorly cytopathic. Strain V4 for example has been shown to be pathogenic iin vitro only to of chicken embryo liver cells. Strain I-2 was reported to produce cytopathic effect (CPE) on chicken embryo kidney (CEK) cells. The present studies confirmed this observation and developed a quantal assay. CEK cells infected with strain I-2 developed CPE characterized by degeneration, rounding, granularity and vacuolation, and the formation of synctia. End points were readily established by microscopic examination of fixed and stained cells. In virus infectivity studies on strain I-2, where multiple titrations are required and where large numbers of samples are used, titration using CEK cell grown in microtitre plates is recommended. Such studies may not be feasible in embryonated eggs.

Improved Statistical Methods for Quantal Assay

Improved Statistical Methods for Quantal Assay

Antiserum to the gp116 glycoprotein of yellow head virus neutralizes infectivity in primary lymphoid organ cells of Penaeus monodon

Yellow head virus (YHV) is an invertebrate nidovirus that has caused mass mortality of cultured Penaeus monodon in Asia. In this study, we investigated whether mouse polyclonal antiserum raised against the YHV gp116 or gp64 structural glycoproteins could neutralize YHV infectivity as determined using an in vitro quantal assay in primary cultures of lymphoid organ cells. Anti-gp116 antiserum showed virus-neutralizing activity whereas anti-gp64 antiserum failed to inhibit infection. The results suggest that gpl16 antiserum blocks binding of virions to cellular receptors to facilitate YHV entry into lymphoid organ cells.

Propagation of infectious yellow head virus particles prior to cytopathic effect in primary lymphoid cell cultures of Penaeus monodon.

Yellow head virus is a highly pathogenic agent that can cause a fatal disease in several species of penaeid shrimps. Using a primary cell culture and an in vitro quantal assay (TCID50), this study sought to determine the propagation profile of yellow head virus after inoculation at a low multiplicity of infection in the lymphoid tissue (oka organ) of Penaeus monodon. Detectable levels of virus were present as early as 24 h post-inoculation. Maximal viral yields were reached by 4 d post-infection, approximately 24 h after the onset of a detectable cytopathic effect, which was normally observable at 3 d post-inoculation. The methodology provides a useful tool for studying yellow head virus-host-cell interactions.

An improved filter elution and cell culture assay procedure for evaluating public groundwater systems for culturable enteroviruses.

Large-scale virus studies of groundwater systems require practical and sensitive procedures for both sample processing and viral assay. Filter adsorption-elution procedures have traditionally been used to process large-volume water samples for viruses. In this study, five filter elution procedures using cartridge filters were evaluated for their effectiveness in processing samples. Of the five procedures tested, the third method, which incorporated two separate beef extract elutions (one being an overnight filter immersion in beef extract), recovered 95% of seeded poliovirus compared with recoveries of 36 to 70% for the other methods. For viral enumeration, an expanded roller bottle quantal assay was evaluated using seeded poliovirus. This cytopathic-based method was considerably more sensitive than the standard plaque assay method. The roller bottle system was more economical than the plaque assay for the evaluation of comparable samples. Using roller bottles required less time and manipulation than the plaque procedure and greatly facilitated the examination of large numbers of samples. The combination of the improved filter elution procedure and the roller bottle assay for viral analysis makes large-scale virus studies of groundwater systems practical. This procedure was subsequently field tested during a groundwater study in which large-volume samples (exceeding 800 L) were processed through the filters.

Development of an in vitro quantal assay in primary cell cultures for a non-occluded baculo-like virus of penaeid shrimp

An in vitro quantal assay (TCID 50) for a non-occluded baculo-like virus isolate from naturally infected Penaeus japonicus obtained from China and experimentally infected P. stylirostris was developed using primary shrimp lymphoid cell cultures in Primaria TM 24-well tissue culture plates. The virus caused cytopathogenic effect (CPE) in the cell cultures as early as 2 day post-infection (p.i.). Initially, the cells rounded up and finally detached from the culture vessel as the infection progressed. At the present time, there is no established quantitative in vitro cell culture protocol for the assay of this baculo-like virus which has been reported by our laboratory to be highly pathogenic for P. stylirostris and P. vannamei, the two species of penaeid shrimp commercially cultured in Hawaii and the Western hemisphere. This quantal assay thus provides a simple and convenient method for the detection and assay of infectious virus in cultured penaeid shrimp.

Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

Asymmetric tolerance distributions: an application of two-piece distributions

A technique is proposed which allows for the fitting of and testing for asymmetric tolerance distributions in quantal assays. The implementation of the technique in statistical packages is discussed and is illustrated by an application to a simple data set.

Analysis of Quantal Response Data.

Data, preliminary analyses and mechanistic models maximum-likelihood fitting of simple models extensions and alternatives extended models for quantal assay data describing time to response overdispersion non-parametric and robust methods design and sequential methods. Appendices: approximation procedures GLMs and GLIM bordering hessians asymptotically equivalent tests of hypotheses computing solutions and comments for selected exercises.

Single- and multi-hit kinetics of immunoglobulin G neutralization of human immunodeficiency virus type 1 by monoclonal antibodies

A quantal assay, based on syncytium formation in the human T cell leukaemia-derived C8166 cell line, was used to determine the kinetics of human immuno-deficiency virus type 1 (HIV-1) strain IIIB neutralization. Three rat monoclonal antibodies (MAbs) were used, under physiological conditions of temperature and antibody concentration. MAb ICR39.3b (IgG2b) neutralized virus with no lag period while the other two MAbs, ICR39.13g (IgG2b) and ICR41.1i (IgG2a), neutralized with lag periods of 5 min and 15 min respectively. It was calculated that the latter two MAbs mediated neutralization by about two and three molecules of IgG per virion respectively. The highest neutralization rate constant (for MAb ICR 41.1i) was over 300-fold less than that of MAbs specific for the haemagglutinin of the enveloped influenza virus type A and for poliovirus type 1.

Analysis of Quantal Response Data

Data, preliminary analyses and mechanistic models maximum-likelihood fitting of simple models extensions and alternatives extended models for quantal assay data describing time to response overdispersion non-parametric and robust methods design and sequential methods. Appendices: approximation procedures GLMs and GLIM bordering hessians asymptotically equivalent tests of hypotheses computing solutions and comments for selected exercises.

Mapping water quality by local scoring

The mean density of bacteria in a water body is commonly monitored using quantal assay. This paper describes the use of local scoring in estimating the spatial distribution of mean density from quantal assay results at a set of point locations. An application to estimating the mean density of fecal coliform bacteria in a coastal pond is presented. Model diagnostics based on a parametric bootstrap are also presented.

Score tests for the single‐hit poisson model in limiting dilution assays

AbstractWhile estimators of the relative frequency of responding cells in limiting dilution assays (LDAs) have been critically evaluated, other statistical issues concerning this quantal assay have not received similar attention. This study investigates LDA validity tests designed to detect deviations from the single‐hit Poisson model. The method employed by Armitage (1959) to detect host variability, or a variable number of false negatives, is extended to other violations of the single‐hit Poisson model. In general, this methodology, based on the general theory of Neyman (1955), is shown to produce asymptotically most powerful and locally most efficient tests for many alternative hypotheses. In addition to composite hypotheses, this method can be extended to produce tests for separate families of hypotheses. These methods are compared with existing LDA validity tests including the weighted regression method of Gart and Weiss (1967), the double‐hit model of Cox (1962), and the general chi‐square goodness‐of‐fit statistic. Monte Carlo simulations show that these score tests have good power and acceptable rejection levels at 0.05 and 0.01. In particular, the score test designed to detect deviations from the Weibull model is shown to produce good power for many alternative models. The chi‐square goodness‐of‐fit statistic, the most widely used LDA validity test, is shown to lack power in detecting many alternative hypotheses.

A Mixture Model for Interval-Censored Time-to-Response Quantal Assay Data

Various mixture models are proposed and fitted to a set of time-to-response quantal assay data. The models provide a natural extension to the time-dependent case of the classical threshold model for quantal assay data. The approach deals simply with interval censoring, and provides flexible families of distributions, for both the mixing probability and the cumulative distribution function for time to response. For data of the kind considered in this paper, mixture models may provide a more suitable description than other models from survival analysis, such as the proportional hazards model.