Double-layer plaque assay for quantification of enteroviruses

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay >or=suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters >or= most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2'-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.     

Initial phase of infection with Tyzzer's organism in cultured mouse hepatocytes

The initial phase of infection with Tyzzer's organisms in cultured mouse hepatocytes was observed using indirect immunofluorescence (IF) and a plaque assay. The organisms adhered poorly to aldehyde- or acetone-fixed cells, but once adhered to host cells, whether methanol-fixed or unfixed, they were not removed by methanol or acetone. By the IF as well as the plaque assay, both of which discriminated intra- and extracellularly located organisms, the number of cell-associated organisms increased linearly up to 3 h post-inoculation. The number of intra-cellular organisms increased rapidly in the first 1 h, followed by linear increase at a much lower rate. Similar results were obtained by the plaque assay.     

Interferon-gamma reduces the sensitivity of cultured and fresh human tumor cells to lysis by lymphokine-activated killer cells

We have shown that interferon-gamma (IFN-gamma), in pharmacologically achievable doses, can reduce the the sensitivity of human tumor cells to lysis by allogeneic lymphokine-activated killer (LAK) effector cells. Cultured tumor cells showed a consistent reduction in sensitivity to lysis following pretreatment for 18 h with 1-10 units/ml IFN-gamma. Tumor cells cultured up to 7 days in 100 units/ml IFN-gamma remained less sensitive to lysis. Induction of protection from LAK did not appear to correlate with IFN-gamma-induced changes in cell growth or proliferation. Reduced LAK sensitivity also did not correlate with the level of expression of major histocompatibility antigens. Eight of 11 surgically obtained human tumor cell specimens showed a reduction in sensitivity to lysis by allogeneic LAK cells following pretreatment with IFN-gamma. IFN-induced reduction of tumor cell sensitivity to lysis by LAK may play a role in altering the host-tumor relationship, since relatively high concentrations of IFN-gamma may exist in the tumor microenvironment.     

Three-Day Assay for Human Cytomegalovirus Applicable to Serum Neutralization Tests

A fluorescing cell assay (FCA) technique utilizing the indirect fluorescent-antibody method to measure human cytomegalovirus (CMV)-infected cells has been applied to the rapid determination of CMV-neutralizing antibody. Human sera with complement fixation titers to CMV of 1/32 or greater and fluorescein-conjugated rabbit anti-human globulin are the primary and secondary reagents in the fluorescent-antibody test. FCA measured in 3 days the same number of infectious units measured by plaque assay in 2 weeks. FCA and plaque assay yielded identical neutralizing antibody titers to CMV in 20 human sera.     

Augmentation of human monocyte-mediated cytolysis by interferon

Human monocytes, separated by either plastic adherence or adherence to microexudatecoated surfaces, from the peripheral blood of most normal donors were shown to have significant cytolytic activity against TU5, a mouse SV40-transformed target cell. Spontaneous cytolysis ranged from 0 to 32% at a 40:1 effector:target (E:T) ratio. Augmentation of cytolysis was usually seen when human fibroblast interferon (IF) (10³–10⁴ units/ml) was cultured with the effector and target cells for the duration of the assay. The mean increase in percentage cytolysis at 40:1 and 20:1 E:T ratios was greater with monocytes obtained by a microexudate method (24.1 and 22.4%) than with monocytes obtained by a plastic adherence method (16.0 and 8.1%). Only a slight augmentation of cytotoxicity was observed when the effector cells were pretreated with IF for 1-hr. The increased levels of cytotoxicity observed when IF was present during the assay did not appear to be due to the toxic effects of IF on the target cells or to a stable increase in the susceptibility of the target cells to lysis.     

The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification

This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages : (1) phage infection of target bacterium ; (2) destruction of exogenous phage ; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml⁻¹ in a time of 4 h and 600 bacteria m⁻¹ for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.     

Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium

Fiala, Milan (University of Washington, Seattle), and George E. Kenny. Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium. J. Bacteriol. 92:1710-1715. 1966.-A reproducible macroplaque assay for six M and three H strains of rhinoviruses has been developed in several human heteroploid cell lines. Plaques were produced only with suitable solidifying agents: purified agar (Ionagar, Agarose) or methylcellulose. Plaque development was greatly enhanced by increasing Mg(+2) to 30 to 40 mm. Diethylaminoethyl (DEAE) dextran also increased plaque sizes, and the effects of Mg(+2) and DEAE dextran were additive. In addition, Ca(+2) substituted for Mg(+2). The suitability of human heteroploid cell lines for rhinovirus plaque assay varied greatly, ranging from insensitivity through partial to complete sensitivity. This assay was six to seven times more sensitive than an end point tube assay. These results indicate that potentiation of plaque formation by Mg(+2) known for some enteroviruses can also be extended to the rhinovirus group of picornaviruses.     

Homologous interference of lymphocytic choriomeningitis virus: detection and measurement of interference focus-forming units.

Lymphocytic choriomeninigitis (LCM) virus defective interfering (DI) particles form foci of protected cells in a monolayer under an agarose-containing overlay medium. Foci originate from one cell dually infected with at least 1 interference focus-forming unit and infectious virus. As a result, an interfering factor is produced and released which interacts with neighboring cells, thereby protecting them against cytopathic lysis by challenge virus. The property of individual LCM virus DI particles to induce countable foci has been made the basis of quantitative assay that is comparable in every respect to the plaque assay of infectious virus and is much more sensitive and probably more accurate than other procedures used to measure LCM virus DI particles. LCM virus was passaged, undiluted, 10 times in cell cultures. When yields were analyzed as to concentrations of PFU and interference focus-forming units, both entities were found to fluctuate with the pattern expected from theoretical considerations.     

Rhinovirus Plaque Formation in WI-38 Cells with Methylcellulose Overlay

A sensitive, reliable plaque assay system is described for five rhinoviruses using freshly prepared methylcellulose overlay and human embryonic diploid cells. Circular plaques with irregular edges, 2 mm in size, were formed by rhinoviruses 1A, 2, 6, and 13 after 6 or 7 days of incubation. A fifth rhinovirus, 17, formed a 1- to 2-mm feather plaque after 14 days of incubation. Plaque counts of rhinoviruses 1A and 13 were not affected by varying the pH of the overlay from 6.9 to 7.5. Plaque sizes and plaque-forming unit values of high passage rhinoviruses 1A and 13 were equivalent when tested at 26, 31, or 36 C. The rhinoviruses tested were sensitive to incubation at 40 C or heating at 50 C. Enhancement of plaques was observed when Mg(++) was incorporated into agar overlays, but enhancement did not occur when Mg(++) was added to methylcellulose overlays.     

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture ^{1, 2}. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages ¹. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay ¹. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques ³.Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay ³, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers ⁴. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID₅₀) ³. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration ⁵. However, its limit of detection is higher compared to a plaque assay ⁴.In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples ^{1, 4, 6}. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample ³.     

Detection of antibodies to Herpesvirus simiae and Herpesvirus hominis in nonhuman primates

Sera from nonhuman primates, predominantly Macaca species, were assayed by a serum neutralization test for antibodies to antigenically related Herpesvirus simiae (B virus) and Herpesvirus hominis type 1. The data indicate that there would have been approximately 50% error in the diagnosis of Herpesvirus simiae infection if these sera had been tested only against Herpesvirus hominis antigen. The role of active guinea pig complement in the serum neutralization test was also evaluated and found to be required by many of the sera for reproducible and enhanced virus neutralization, particularly for B virus antibody determination. A plaque reduction assay was found to be highly sensitive, especially when complement (2.5-5.0 hemolytic units) was added, but impractical for large-scale serum surveys.     

Evaluation of cell lines and immunofluorescence and plaque assay procedures for quantifying reoviruses in sewage.

Twelve continuous cell lines were tested to determine their sensitivity to reovirus types 1, 2, and 3 isolated from sewage. Madin-Darby bovine kidney (MDBK), rhesus monkey kidney (LLC-MK2), and human embryonic intestinal (intestinal 407) cells were most sensitive, respectively. In a similar study, MDBK cells were more sensitive than LLC-MK2 and Buffalo green monkey kidney (BGM) cells to sewage-isolated, protamine-precipitated reoviruses which had not been serotyped and had no previous cell contact. Sewage-isolated, protamine-precipitated reoviruses were also used in conjunction with MDBK cells in a comparative evaluation of immunofluorescent cell count and plaque assay procedures. The immunofluorescence assay is more sensitive and more rapid than the plaque assay. Reoviruses in excess of 10(4)/liter of raw sewage were detected by the immunofluorescent cell count assay.     

In vitro purging of clonogenic leukaemic cells from human bone marrow by interferon-γ-activated monocytes

With a view to the immunologically mediated purging of autologous bone marrow transplants in acute myeloid leukaemia, the efficacy of cytotoxic monocytes to cradicate leukaemic cells has been studied using clonogenic assays. U937 cells were found to be sensitive to highly purified and interferon--activated human monocytes whereas HL60 cells were rather resistant as measured in an MTT-based cytotoxicity assay under liquid conditions. A spectrophotometric clonogenic assay measured almost complete inhibition of clonogenic activity for U937 cells at low effector-to-target cell (E/T) ratios of at least 0.1. Limiting dilution analysis detected a 2–3 log₁₀ unit reduction in clonogenic activity. In an experimental mixture of U937 cells with a 20-fold excess of normal bone marrow nuclear cells a maximum 2-log₁₀-unit killing could be measured at E/T=10. Only at high E/T ratios could a reduction in granulocyte/macrophage-colony-forming units (cfu) be observed with only marginal effects on erythroid cfu and erythroid burst-forming. In conclusion, cytotoxic monocytes are highly potent anti-leukaemic effector cells, as measured in clonogenic assays, that do not compromise normal human progenitors.     

Mitochondrial inhibitory factor 1 (IF1) is present in human serum and is positively correlated with HDL-cholesterol

Background

Mitochondrial ATP synthase is expressed as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein component in High Density Lipoproteins (HDL). On hepatocytes, apoA-I binds to cell surface ATP synthase (namely ecto-F₁-ATPase) and stimulates its ATPase activity, generating extracellular ADP. This production of extracellular ADP activates a P2Y₁₃-mediated HDL endocytosis pathway. Conversely, exogenous IF1, classically known as a natural mitochondrial specific inhibitor of F₁-ATPase activity, inhibits ecto-F₁-ATPase activity and decreases HDL endocytosis by both human hepatocytes and perfused rat liver.

Methodology/Principal Findings

Since recent reports also described the presence of IF1 at the plasma membrane of different cell types, we investigated whether IF1 is present in the systemic circulation in humans. We first unambiguously detected IF1 in human serum by immunoprecipitation and mass spectrometry. We then set up a competitive ELISA assay in order to quantify its level in human serum. Analyses of IF1 levels in 100 normolipemic male subjects evidenced a normal distribution, with a median value of 0.49 µg/mL and a 95% confidence interval of 0.22–0.82 µg/mL. Correlations between IF1 levels and serum lipid levels demonstrated that serum IF1 levels are positively correlated with HDL-cholesterol and negatively with triglycerides (TG).

Conclusions/Significance

Altogether, these data support the view that, in humans, circulating IF1 might affect HDL levels by inhibiting hepatic HDL uptake and also impact TG metabolism.     

The development of a peptide SRM-based tandem mass spectrometry assay for prenatal screening of Down syndrome

Two new biomarkers, serum amyloid-P (SAP) and plasma C1-inhibitor protein are elevated in the maternal circulation of mothers carrying Down syndrome foetuses. Much emphasis of late\ has been put on the lack of translational tests being developed following the identification of new biomarkers. We have created a single-reaction-monitoring (SRM) tandem mass spectrometry-based assay for the quantitation of these biomarkers and compared these results with an in-house developed immunofluorescence-based technique (IF). This MS-based assay is a rapid 5 min test and a simple "one pot reaction," requiring only 5μl of plasma. To evaluate the potential of SRM-based quantitation in a clinical setting, SAP and C1-inhibitor were quantitated in 38 normal and Down syndrome affected pregnancies. Plasma SAP levels in the Down's group were significantly raised at 10-14 weeks (p<0.0015) and 14-20 weeks (p<0.0001). Plasma C1-inhibitor levels were also observed significantly elevated in the Down's group (10-14 weeks, p<0.0193, 14-20 weeks, p<0.0001). Analysis using the IF technique did not show any significant elevation of plasma SAP levels or C1-inhibitor levels. This rapid and sensitive assay demonstrates the potential of multiplexed tandem MS-based quantitation of proteins in chemical pathology labs and in a more cost-effective, accurate manner than conventionally used antibody methods.     

Evaluation of consistency in quantification of gene copy number by real‐time reverse transcription quantitative polymerase chain reaction and virus titer by plaque‐forming assay for human respiratory syncytial virus

The plaque‐forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque‐forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT‐qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque‐forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT‐qPCR. Two real‐time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque‐forming units. In conclusion, RT‐qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque‐forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

     

Fluorescent measurement of desmin intermediate filament assembly

Intermediate filaments (IF) are cytoskeletal elements that are believed to play a major role in the specification and maintenance of cell form. Although previously thought to be stable and static because of their relative insolubility in physiological solvents, IF have recently been shown to have dynamic properties not unlike those of other cytoskeletal elements. The methodology for measuring this dynamic behavior, however, has been mostly borrowed from studies of other filament proteins and are poorly suited to IF because of their unusual physicochemical properties. In this report we introduce a fluorescence assay for quantifying in vitro IF assembly. Desmin subunits labeled with iodoacetamidofluorescein (IAF) to approximately 0.4 mol/mol retain the ability to polymerize into filaments indistinguishable from unlabeled IF in the electron microscope. By spectrophotometry, however, up to 90% of the starting fluorescence is quenched upon maximal IF assembly from IAF-desmin subunits. This quench is proportional to the total concentration of desmin subunits and is a sensitive measure of the assembly process. The critical concentration of assembly, measured at 170 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.0, is 0.2 microM. This indicates that a significant level of unpolymerized desmin exists in steady-state equilibrium with polymerized filaments under these conditions and suggests that IF subunit-filament equilibria may play a role in cytoskeletal dynamics.     

应用明胶凝集颗粒试验检测人群中T细胞白血病病毒抗体

曾毅等报道,应用间接免疫荧光法(IIF)检测了8,279份正常成年人血清标本,发现3例T细胞白血病病毒(HTLV)抗体阳性:一例马杨氏,丈夫是日本人(HTLV抗体也阳性),侨居南京46年;第二例是台湾籍妇女;第三例是位侨居北京的日本人。650份各类白血病病人血清中,一例成人T细胞白血病患者HTLV抗体阳性,此人是船员,常去日本。     

Double-Layer Plaque Assay for Quantification of Enteroviruses

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay ≥ suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters ≥ most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2′-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.     

Large scale production of human lymphoblastoid (Namalva) interferon

Summary Large scale production of human lymphoblastoid (Namalva) interferon (IF) is described. Cell propagation, in up to 50 1 culture volume, was carried out in a low cost medium by a semi-continuous cultivation method. IF was induced by Sendai virus, testing two induction methods. The yield of crude IF varied in the range of 12 – 100 × 10³ IF units.ml^-1. A weekly production output of 1 – 5 × 10⁸ units crude IF was obtained.