Improved plaque assays for Rickettsia prowazekii in Vero 76 cells.

Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 microgram of emetine per ml and 20 micrograms of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics.     

Titration of avirulent Newcastle disease virus by the plaque assay method

Parameters of a plaque assay for avirulent strains of Newcastle disease virus (NDV) were optimized for reproducibility and optimum titer in LLC-MK₂ cells. Plaques were visible after 2 days and maximum virus titers were reached in 3 days. Virus titers were not affected by continued incubation through 6 days, although plaque size increased. An adsorption volume of 0.1 or 0.2 ml per 60 mm Petri dish was optimal, as was an adsorption time of 45 min. Trypsin (2.5 μg/ml) and magnesium sulfate (0.03 M) were essential requirements of the overlay medium and the presence of DEAE-dextran (0.02%) resulted in a 30% increase in titer. The use of cell monolayers, 1, 2, or 3 days old facilitated the performance of multiple assays per week and did not affect the virus titer.     

Sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus.

A sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus was developed in Vero and MA 104 cell cultures. The tests were performed in semimicrotoiter trays containing 24 wells, 16 mm in diameter. Parainfluenza virus type 1 formed plaques in Vero and MA 104 cells only when trypsin was added to the overlay medium. Plaquing of parainfluenza virus type 1 was more sensitive and technically reproducible in MA 104 cells than in Vero cells. Parainfluenza virus types 2 and 3 and respiratory syncytial virus readily formed plaques in Vero cells. Plaques with all viruses were necrotic in character, except for plaques produced by parainfluenza virus type 3, which appeared red due to an increased uptake of neutral red by infected cells. Different conditions for plaquing of the four viruses had to be used to obtain plaques of suitable size. Antibody titers of commercially prepared guinea pig typing sera were 5- to 50-fold higher by the plaque neutralization test than by complement fixation. The addition of guinea pig immunoglobulin G antiglobulin to the serum-virus mixtures enhanced the conventional neutralization test 5- to 10-fold. The sensitivity and specificity of the plaque neutralization test was also determined with sera of marmosets experimentally infected with parainfluenza virus types 1 and 3. The generally low postinfection titers could be enhanced, on the average, 40-fold by using human immunoglobulin G antiglobulin in the neutralization test. A low degree of cross-reactivity was shown between parainfluenza virus types 1 and 3 both in the conventional neutralization test and in the anti-immunoglobulin enhanced neutralization test.     

Plaque assay for avian metapneumovirus using a Japanese quail fibrosarcoma cell line (QT-35)

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40,000 cells per cm(2) when using a 1.5% CMC overlay or 100,000 cells per cm(2) when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.     

An improvement in the plaque assay of rabies virus in chick embryo cells

Summary The faint plaques formed with HEP Flury strain of rabies virus in chick embryo cells by the original method were thought ascribable to acid production from virus-infected cells coupled by the weak buffering action of the agar overlay medium. When the overlay medium contained appropriate concentrations of alkalies, clear plaques could be observed. The optimal conditions were (i) incorporation of 0.02% NaOH, 0.0025m Tris-HCl buffer of pH 8.2 and 2% calf serum in the base overlay medium, and (ii) neutral red staining after 7 days' incubation at 35 C. Under these conditions the plaque size was approximately 2 mm in diameter and autointerference caused by higher multiplicity of infection showed a diminishing trend. Mouse passaged NOPM strain also formed fairly clear plaques when agarose was substituted for agar, the plaque titer being almost equal to its mouse LD titer. That these plaques were formed by infection of the virus was testified by a neutralization test using a standard antiserum prepared with rabbit passaged CVS strain. Adsorption of rabies virus to chick embryo cells was found to proceed slowly, the maximal adsorption requiring 4 hours or longer. The adsorption efficiency was equal between 35 and 37C.     

Differential effect of limitation in oxygen supply on plaquing and multiplication of adenovirus and poliovirus

Summary Adenovirus and poliovirus were grown and plaqued in KB cell sheet cultures under nutrient agar medium in Petri dishes in a desiccator continuously flushed with atmospheres containing O₂ at various concentrations. Virus multiplication and plaquing were determined in the central half of the culture, where the depth of the agar overlay was uniform.Under 3 percent O₂ relative to the control under 19 percent O₂, adenovirus plating efficiency was <1 percent in the central half, and the average diameter of the few plaques discernible there was only 20 percent of the average control plaque diameter, whereas relative plating efficiency of poliovirus did not differ significantly from 100 percent and average plaque diameter under 3 percent O₂ was 60 percent of average control plaque diameter.Multiplication curves determined under 3, 5, 10 and 19 percent O₂ for each virus at total input of 10,000 PFU per cell sheet showed that the capacity of poliovirus to multiply under the hypoaerobic conditions of 3 and 5 percent O₂ was much stronger than that of adenovirus. Under these conditions, poliovirus completed its eclipse phase in <0.5 day and multiplied to high titer, whereas adenovirus was still in eclipse 5 days postinoculation.With 2 Figures     

Studies of laryngotracheitis virus in avian tissue cultures II. Deficient adsorption of a strain of laryngotracheitis virus

Summary In the plaque assay of the N71851 strain of laryngotracheitis virus (LTV) in chicken kidney (CK) monolayer cultures, the number of plaques which developed depended upon the treatment between adsorption and addition of agar overlay medium. Cultures in which the inoculum was aspirated after the adsorption period had a greater number of plaques than cultures in which the inoculum was aspirated and the cultures were washed. This effect was found to be due to a deficiency of virus to adsorb to cells. Under the conditions of the assay, the efficiency with which virus adsorbed to monolayers was too low to be measured. Therefore, the efficiency of the assay was also low. The initial association between virus and cells was easily disrupted by washing when adsorption was carried out at 5°C to prevent penetration. Treatment of cultures with diethylaminoethyl-dextran for one hour prior to inoculation increased the number of plaques in treated compared with untreated cultures by a factor of five.     

A simplified plaque assay for respiratory syncytial virus--direct visualization of plaques without immunostaining

Due to their generally small size, detection of respiratory syncytial virus (RSV) plaques commonly relies on immunostaining using either polyclonal antisera or a pool of monoclonal antibodies against the surface fusion glycoprotein. Disadvantages include the costs of the antibodies and substrate, and microscopic examination is often needed for counting of plaques. By optimizing many parameters, greatly improved plaque assays for RSV A2, Long and RSV B 18537 strains using Vero or HEp-2 cells have been developed. Although many groups use Vero cells for plaquing, after optimization plaques were much larger in HEp-2 cells. Both cell types needed to be confluent at infection and agarose was more suitable than carboxymethyl cellulose for the overlay. Overlay medium for HEp-2 was DMEM/F12, 0.3% agarose, and for Veros it was Liebovitz L15, 5% fetal calf serum, 0.3-0.5% agarose. After 5-7 days, monolayers were fixed with 1% formal saline overnight, agarose was removed and monolayers were stained with 0.05% neutral red in water. Plaques were readily visible by eye and plates could be dried and stored permanently. Parainfluenza virus type 3 also formed large plaques in HEp-2 cells under the conditions optimal for RSV.     

A Fluorescent micro-plaque assay for hog cholera virus

Summary A plaque assay system was investigated for the purpose of enumerating infective units of the noncytopathogenic Ames strain of hog cholera virus. In the absence of apparent cytologic changes, discrete foci of infection were made evident by fluorescent antibody staining. The localization of primary plaques was accomplished by the incorporation of anti-hog cholera serum into the nutrient medium of infected cell cultures. Plaque size was directly related to incubation time and inversely related to the concentration of antiserum. It was concluded that the primary mechanism for the spread of infection in cell culture is through the dissemination of extracellular hog cholera virus. However, it was also noted that a concentration of antiserum in excess of that required to prevent the formation of secondary plaques still allowed contiguous peripheral extension of primary plaques; thus the possibility of direct cell-to-cell transfer of infectious virus was suggested.The reliability of the fluorescent plaque assay was confirmed by the following observations. In the presence of a sufficient concentration of antiserum, the number of plaques did not increase with continued incubation of infected cultures. In addition, a linear relationship was found between hog cholera virus concentration and plaque counts, and, finally, titers obtained by the plaque assay were essentially the same as 50% cell culture infective dose titers.     

Identification of two plaque variants of Guaroa virus

Summary The prototype CoH35211 strain of Guaroa virus contained 2 different plaque populations when French or Mann agarose were used for solidifying the overlay medium. The pl1L (large) and p12S (small) plaque variants were similar with respect to pathogenicity for suckling mice (SM), titers achieved in SM brains after intracranial inoculation and time of appearance of plaques under agarose overlay. The prototype CoH35211 strain and the 2 plaque variants were indistinguishable by complement-fixation and plaque-neutralization tests. However, the 2 plaque variants differed with respect to plaque size and sensitivity to the inhibitory effects of sulfated agar polysaccharides. The only different serological relationship was a one-way cross-reaction between p12S antibody and California encephalitis and Tahyna viruses by plaque-neutralization test.     

Reproducible plaquing system for rhinovirus serotypes in HeLa cells--agarose suspension.

HeLa cell--agarose suspension method provides an excellent plaquing system for assay of rhinovirus serotypes which ordinarily produce indistinct plaques on cell monolayers. The variable plaque size of cloned virus progeny does not involve antigenic change in phenotype as shown by plaque neutralization test. The system has proved useful for accurate and reproducible plaque assay of rhinoviruses, assay of serum neutralizing antibodies and cloning of viruses. The plaques produced are large, clear and reproducible. The factors which influence the plaquing of rhinoviruses in this system are the age of the cells, cell density and pH of the medium.     

A plaque assay system for rinderpest virus and its use in characterising virus adsorption

Summary A continuous line of calf kidney cells was found suitable for the production of plaques by five strains of rinderpest virus known to differ in their virulence for cattle. No differences were seen in the morphology of the plaques produced by these strains. Plaques were first seen after 6 days incubation, and if neutral red was incorporated in the overlay appeared as red centres composed of a small cluster of cells taking up this stain more strongly than the healthy background cells; thereafter plaques enlarged very slowly and were only 1.5–2.0 mm in diameter after 14 days. Plaque counts did not increase beyond the 6th day of incubation and were unaffected by either agarose concentrations of 0.4 to 1.5% or overlay pH values between 6.7 and 7.85. It was shown that neutral red included in the initial overlay did not affect the sensitivity of virus titrations.Adsorption of rinderpest virus proceeded equally well from a number of different suspending fluids but the rate of adsorption was closely related to the volume of diluent in which the virus was suspended. At 37° C adsorption ceased after approximately 30 minutes exposure, while at either 4° or 22° C relatively less virus was adsorbed and reactions were completed after 10 minutes. It was not possible to show clearly the dependence of adsorption on pH as the stability of the virus at 37°C evidently varied with different pH values.     

Antiviral neutralizing antibody to Hantaan virus as determined by plaque reduction technique

Summary The 76–118 strain of Hantaan virus was titrated in E6 (Vero) cells by the plaque method using agarose overlay medium. Visible plaques, formed 10 days post-infection, were uniformly 2–3 mm in diameter. Dose-response experiments showed that a single infectious particle initiated the formation of a plaque. Infectivity titers by the plaque method were equivalent to those obtained by the endpoint method (TCID₅₀) using the immunofluorescence antibody technique (IFA) for antigen detection. The single-cycle growth pattern of the virus showed an eclipse phase of 7 to 9 hours, with production of cell-free infectious virus 18 hours post-infection. Plaque reduction neutralization tests suggested that complement enhanced the neutralizing activity of sera; rat sera were particularly complement-dependent. The plaque reduction neutralization test was about 10 times more sensitive than the TCID₅₀ neutralization test. Convalescent phase sera from patients with hemorrhagic fever with renal syndrome (HFRS) having higher IF antibody titers to Hantaan virus than to nephropathia epidemica (NE) virus were capable of neutralizing Hantaan virus, while sera from patients with higher IF antibody titers to NE virus than Hantaan virus did not contain neutralizing antibody to Hantaan virus.With 3 FiguresOn leave from the Department of Virology, Kyushu University School of Medicine, Fukuoka 812, Japan.     

Use of gum tragacanth overlay, applied at room temperature, in the plaque assay of fish and other animal viruses.

Fish cells derived from rainbow trout gonad or Atlantic salmon are sometimes damaged by the relatively high temperature of agar overlay widely used for plaquing animal viruses. This heat-induced cell damage can be avoided by the use of gum tragacanth, which may be applied at room temperature. When the medium was buffered with tris(hydroxymethyl)aminomethane-hydrochloride and NaHCO3, the plaque assay could be performed without the use of a CO2 incubator. Using this method, a number of animal viruses were plaqued on a variety of cell monolayers at different temperatures under atmospheric ocnditions.     

Enhanced interferon production from chick embryo cells aged in in vitro

The plaque-forming efficiency of Sindbis virus decreased as much as 1000-fold, and plaque size was diminished markedly, when tested on chick embryo cell monolayers aged in vitro. The plaquing efficiency and plaque size of Newcastle disease virus was unaffected. The reaction(s) associated with aging in vitro which lead to lowered plaquing efficiency are slowed considerably when cell monolayers are: (1) established in Simpson-Hirst medium rather than standard growth medium; (2) held at 31 degrees ; (3) given a regimen of daily medium changes; or (4) trypsinized and used as young secondary cultures. A loss in the average yield of virus per cell accompanies the loss in plaquing efficiency of Sindbis virus on aged monolayers. Adding actinomycin D to the aged cells at the time of infection eliminated completely the inhibition of Sindbis virus replication. Cells aged for 7 days in vitro were found to produce up to 32 times more interferon than cells 1-2 days old and were more sensitive to the action of interferon. The decrease in efficiency of Sindbis virus plaquing and yield in aged cells is accounted for by their development of an enhanced capacity to synthesize interferon upon appropriate stimulation. The process of contact inhibition and its concomitant regulation of macromolecular synthesis seems implicated in the aging phenomenon in that it may produce a generalized state of "enhanced derepressibility" in the cell.     

Variables affecting viral plaque formation in microculture plaque assays using homologous antibody in a liquid overlay.

A liquid antibody microculture plaque assay and the variables that govern its effectiveness are described. The assay is based on the principle that low concentrations of homologous antibody can inhibit secondary plaque formation without inhibiting formation of primary plaques. Thus, clear plaques that followed a linear dose response were produced. The assay was found to be more rapid, less cumbersome, and less expensive than assays using agar overlays and larger tissue culture plates. It was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique in measuring infectious coxsackievirus type B-3. It was more sensitive in assaying adenovirus type 3 and Western equine encephalomyelitis, vesicular stomatitis, Semliki forest, Sendai, Sindbis, and Newcastle disease viruses than were liquid, carboxymethylcellulose, and methylcellulose microculture plaque assays. The variables influencing sensitivity and accuracy, as determined by using coxsackievirus type B-3, were: (i) the inoculum volume of virus; (ii) the incubation period of virus; and (iii) the incubation temperature.     

Plaque Formation by Rickettsia conori in WI-38, DBS-FRhL-2, L-929, HeLa, and Chicken Embryo Cells

Mammalian cells particularly suitable for the study of specialized aspects of rickettsial biology were tested for their ability to support plaque formation by Rickettsia conori. The detection of plaques was substantially influenced by the combination of growth medium and cell type used. Large plaques (2.0 to 3.0 mm in diameter) occurred by 8 days postinfection in WI-38 and DBS-FRhL-2 cells supported by medium 199. Smaller plaques (0.5 to 1.0 mm in diameter) were seen in L-929 and HeLa cells at 8 to 11 days postinfection and were more discernible in cells supported with Eagle minimal essential medium. Chicken embryo cells maintained in Dulbecco's modified Eagle medium exhibited large spherical plaques with a diameter of approximately 1.5 mm by 8 days postinfection.     

Plaque assay of variola virus in a cynomolgus monkey kidney cell line

Summary Variola virus produced plaques in a cynomolgus monkey kidney cell line, JINET, under agar overlay medium. The plaque morphology of variola virus in these cells was clearly distinct from that of other members of the variola-vaccinia subgroup of poxvirus and the plaque morphology marker of variola virus may be used for the identification of variola virus. The plaque number of variola virus was enhanced by the addition of DEAE-dextran and MgCl₂ to agar overlay medium at concentrations of 100 to 200 g/ml and 15 to 45 mm, respectively. The sensitivity of the plaque method in the presence of 100 g/ml of DEAE-dextran and 25 mm MgCl₂ in the overlay medium was equal to that of the usual CAM method.     

Plaque formation by virulent Shigella flexneri.

An in vitro tissue culture plaque assay was developed to investigate the intracellular replication and intercellular spread of virulent shigellae. Shigella plaques were formed in HeLa cell monolayers in the presence of an agarose overlay containing tissue culture medium and gentamicin, which eliminated extracellular bacterial growth. Microscopically, the plaques were characterized by a central area of dead host cells surrounded by cells infected with shigellae. Cells further away from the plaque center were uninfected. Inclusion of chloramphenicol or nalidixic acid in the overlay completely abolished plaque formation. Plaque formation was completely inhibited when infected monolayers were shifted from 37 to 30 degrees C. Shifting infected monolayers from 30 degrees C, where plaques do not form, to 37 degrees C resulted in the formation of plaques. Cultures of Shigella boydii, Shigella sonnei (form I), and all six serotypes of Shigella flexneri produced plaques. Shigellae isolated from plaques were Sereny test positive, contained a 140-megadalton plasmid, and were gentamicin sensitive. Noninvasive shigellae did not form plaques.     

Development of dengue virus plaques under serum-free overlay medium.

An improved plaque assay for dengue virus was developed utilizing baby hamster kidney (BHK-21) cells initially grown in shaker culture. Different media preparations were tested for uniform and fast formation of BHK-21 cell sheets. Several overlay formulas were tested to develop a rapid plaque assay in 6- and 24-well plastic plates. The best results were obtained utilizing Eagle minimal essential medium (pH 7.2 to 7.4) supplemented with 1 mg of NaHCO3 per ml and 5% newborn calf serum for the formation of cell monolayers after 8 to 24 h of incubation at 37 degrees C. Serum-free Eagle minimal essential medium supplemented with 1% methylcellulose and buffered with 10 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (pH 7.4 to 7.6) was used as an overlay medium. This system allowed for plaque formation after 3 days of incubation of dengue type 2 virus and after 4 days for dengue type 1 and 4 viruses.