Infectivity assays for the Autographa californica nuclear polyhedrosis virus using Spodoptera littoralis cells

Tissue culture ID50 and plaque assays for the Autographa californica nuclear polyhedrosis virus, using the Spodoptera littoralis cell line HPB-SL, were developed. Direct comparison using these assay methods showed that these cells were as susceptible to infection as the more commonly used Spodoptera frugiperda cell line IPLB-SF-21. Both infectious tissue culture supernatants or virus isolated directly from polyhedra could be titrated. It was important to use low cell seeding densities in the assays so that clear centres of infection formed. Dose-response curves indicated that one infectious particle was capable of initiating an infection. Virus could be cloned using either method even though, for the plaque assay, plates had been stained. The tissue culture ID50 assay was performed using 96-well plates and required an incubation period of about 10 days. The plaque assay used a simple nutrient agarose overlay and an incubation period of 5-6 days. Easily countable plaques of 0.3-1.2 mm diameter were detected after staining with iodonitrote-trazolium chloride. The plaques comprised areas of inhibited cell division and round or dead cells. Most plaques contained only some cells with polyhedra and yields averaged about 1/cell. Occasionally plaques or infected wells were found in which no polyhedra could be seen. These infectivity assays are therefore not dependent on polyhedra formation.     

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.     

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.     

Plaque formation by avian infectious bronchitis virus in primary chick embryo fibroblast cells in the presence of trypsin

Summary Ten strains of avian infectious bronchitis virus (IBV) were titrated as plaqueforming units in primary chick embryo fibroblast cells. In the absence of trypsin, plaques were only formed by Beaudette-42 and Iowa-609 strains. When trypsin was incorporated in the overlay medium of cell monolayers, all the IBV strains tested produced plaques within 4 days after inoculation. Incorporation of 20–40 µg of trypsin per ml of the overlay medium seemed to be suitable for plaque formation of IBV. A preliminary investigation was made of the mode of action of trypsin.With 2 Figures     

Evaluation of a Japanese quail fibrosarcoma cell line (QT-35) for use in the propagation and detection of metapneumovirus

A Japanese quail fibrosarcoma cell line (QT-35) was evaluated and compared to Vero cells for its utility in metapneumovirus propagation, titration and serological detection by indirect immunofluorescence staining. Cell characteristics such as growth kinetics at different passage levels and seeding density in 96-well plates using various media formulations were studied in order to determine suitable assay parameters. Specifically, QT-35 cells supported the replication of a subgroup A metapneumovirus, strain 14/1, when maintained in DMEM containing a high level of glucose (4500 mg/l) and 2% gamma-irradiated fetal bovine serum (gamma-FBS). There appeared to be a decreased ability of metapneumovirus produced in chicken embryo fibroblast (CEF) cells to replicate to high titers in QT-35 cells, however, this apparent restriction was overcome after the second passage resulting in high titered stock. Metapneumovirus produced in Vero cells and propagated in QT-35 cells produced high titered stock after the first passage. Viral titers determined in Vero and QT-35 cells were comparable, when the latter cell line was used at passage levels < or = 20 and seeded between 5.0 x 10(4) and 1.0 x 10(5) cells/0.33 cm(2) in hgDMEM containing 10% gamma-FBS, with a reduction to 2% gamma-FBS when the virus was applied to the cell monolayers 24 h post-seeding. After infection with metapneumovirus, QT-35 cells exhibited syncytia, similar to those in metapneumovirus-infected Vero cells, which were readily detected by indirect immunofluorescent (IF) staining.     

Optimal conditions for titration of SV40 by the plaque assay method

The parameters of the Simian Virus 40 (SV40) plaque assay on African green monkey kidney cells were optimized for reproducibility and maximum plaquing efficiency. Plaques were visible as early as 8 days postinfection; maximum titers were obtained with a 10- to 11-day incubation period. Titers read 12-16 days postinfection were not significantly higher than those observed after 10-11 days. Adsorption volumes greater than 0.1 ml/60 mm Petri dish decreased plaque forming units (PFUs) detected. Times greater than 60 min for adsorption of virus to the cell monolayer did not significantly increase the titer; adsorption times less than 60 min resulted in decreased titers. Under standard conditions, 3 ml of overlay medium containing 0.8% agar was applied following virus adsorption and again on days 5 and 10. Concentrations of fetal calf serum (FCS) in the overlay medium of 2.5 to 7.5% gave equal plaque formation. FCS concentrations of 1 and 10% resulted in slightly decreased and increased plaquing efficiencies respectively. Of the reagents tested, agar or agarose containing overlay media produced plaques of maximum number and size. An overlay of methyl cellulose resulted in the same number of plaques, but their size was reduced by approximately 70% relative to those observed in agar; thus longer incubation times were required. Gum tragacanth overlay medium was actually inhibitory to plaque development. DEAE-dextran, dextran sulfate, or DMSO added to agar overlay medium did not enhance plaque number or size, nor did they shorten the incubation period required for their detection.     

Cytopathology, plaque assay, and heat inactivation of hepatitis A virus strain HM175

Hepatitis A virus (HAV) strain HM175 derived after repeated subculture of persistently infected B-SC-1 cells caused a specific cytopathic effect upon acute infection of B-SC-1 cells. The virus formed visible plaques on B-SC-1 cell monolayers after 9 to 14 days of incubation at 34 degrees C, and virus can therefore be titrated by plaque assay. Virus could be re-isolated from plaques of infected cells, which allows the clonal isolation of HAV variants. The stability of a plaque-purified variant of HAV at elevated temperatures exceeded that of poliovirus type 1, but this variant is less stable than previously reported strains of HAV.     

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.     

Development and optimization of a direct plaque assay for human and avian metapneumoviruses

The genus Metapneumovirus within the subfamily Pneumovirinae and family Paramyxoviridae includes only two viruses, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), which cause respiratory disease in humans and birds, respectively. These two viruses grow poorly in cell culture and other quantitation methods, such as indirect immuno-staining and immuno-fluorescent assays, are expensive, time consuming, and do not allow for plaque purification of the virus. In order to enhance research efforts for studying these two viruses, a direct plaque assay for both hMPV and aMPV has been developed. By optimizing the chemical components of the agarose overlay, it was found that both hMPV with a trypsin-independent F cleavage site and aMPV formed clear and countable plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) after 5 days of incubation. The plaque forming assay has similar sensitivity and reliability as the currently used immunological methods for viral quantitation. The plaque assay is also a more simple, rapid, and economical method compared to immunological assays, and in addition allows for plaque purification of the viruses. The direct plaque assay will be a valuable method for the quantitation and evaluation of the biological properties of some metapneumoviruses.     

Adaptation of the plaque assay methodology for dengue virus infected HepG2 cells

The HepG2 cell line is a useful tool for studying dengue virus-cell interactions but as it grows in clumps rather than monolayers, it does not readily adapt itself to the standard plaque assay technique. We therefore sought to develop an indirect plaque assay methodology. Initially HepG2 cells were infected with dengue virus serotype 2 and post-infection incubated for between 0 and 16 h before being treated with trypsin to separate the cells, followed by dilution and plating onto pre-grown monolayers of Vero cells in six well plates. After 7 days incubation and crystal violet staining, plaques were observed at all time points, although there was a relationship between number of plaques and post-infection incubation time, with the longest post-infection incubation time giving the highest number of plaques. To validate the assay with respect to virus input, the experiment was repeated at both the 0 and 16 h post-infection incubation times with different virus: cell levels. At both post-infection incubation times the response of input virus to plaque number was linear. This is a useful adaptation of the plaque assay methodology and one that may be applicable to other virus/cell line combinations.     

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.     

A simple plaque assay for influenza A2 viruses and its application to antiviral screening

Summary Influenza A₂/Singapore and A₂/Hong Kong virus strains formed plaques in monolayers of monkey kidney and dog kidney cells. Using a serum-free overlay containing Earle's balanced salt solution with 1% lactalbumin hydrolysate, 1.4 g/1 sodium bicarbonate and 0.6% agarose, plaque formation occurred in 4–7 days. By incorporating compound in the overlay, plaque inhibition was demonstrated by Amantadine, Rimantadine and the isoquinoline derivatives designated Pfizer UK-2054, UK-2617, UK-2762 and UK-2888.     

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.     

Parvovirus (feline panleucopaenia virus) plaque formation

Summary A plaque assay was developed for feline parvovirus (FPV; feline panleucopaenia virus) in a feline embryo (FEmb) cell line. Higher numbers and larger diameter plaques were obtained with a) seeding rates of 0.7 × 10⁵ and 1.5×10⁵ cells cf. 3×10⁵ and 6×10⁵ cells/well of 35 mm diameter, b) synchronised cells infected at the G₁—S interface cf. nonsynchronised cells and c) 5 to 6 days incubation post inoculation.The plaque assay was standardised by using serum deprivation for 24 hours to synchronize cells, a seeding rate of 1.5×10⁵ cells/35 mm diameter well, inoculation of virus 16 hours post seeding followed by 5 days incubation. The standardised assay gave consistent, reproducible results. A dose-response curve using the assay showed a linear, 45° slope, relationship between plaque forming units and virus dilution which further verified the sensitivity and reliability of the assay. Plaques produced by wild type and plaque purified virus were inavriably non uniform in diameter; diameter of plaques in fact followed a normal frequency distribution under standard assay conditions.With 3 Figures     

A quantitative comet assay: imaging and analysis of virus plaques formed with a liquid overlay

Although the plaque assay defines a "gold-standard" for measuring virus infectivity, its reliance on plaque counting limits its sensitivity. When the assay is performed with a liquid overlay, instead of agar overlay, spontaneous flows can promote a uni-directional spread of infection, creating elongated regions of cytopathology that resemble comets. As a model system comet and plaque cultures of vesicular stomatitis virus (VSV) on baby hamster kidney (BHK-21) cells were compared. Host-cell monolayers were infected with VSV particles, incubated 15 h in the presence of liquid or agar overlays and stained. VSV formed significantly larger comets than plaques, consistent with a mechanism of flow-enhanced spread. When antiviral drug (5-fluorouracil) was incorporated into the liquid overlay, comet sizes were reduced in a dose-dependent manner. Images of infected monolayers, acquired using a simple digital scanner, enabled a quantification of the inhibitory effect of the drug on infectivity. The resulting measure of drug susceptibility was found to be 18-fold more sensitive than the IC(50) measure attained by the traditional plaque-reduction assay. This quantitative comet assay has the potential to similarly enhance the sensitivity of infection measures for other plaque-forming viruses.     

绿色荧光标记重组人偏肺病毒空斑形成滴度测定方法的建立

目的 建立用于绿色荧光标记的重组人偏肺病毒(GFP-rhMPV)空斑形成滴度测定方法.方法 基因组中插入绿色荧光蛋白基因的hMPV全序列cDNA质粒和主要蛋白质表达质粒转入细胞293T后获得感染性重组hMPV.GFP-rhMPV在Vero-E6细胞中连续传代提升病毒滴度并保存.将等倍稀释的重组病毒液接种常规制备的Vero-E6细胞单层,用含或不含胰酶的低熔点琼脂精凝胶覆盖细胞,孵育一定时间后,采用荧光显微镜下计数荧光空斑数和抗原抗体蓝斑形成法计算病毒滴度.结果 感染后3 d,荧光显微镜下GFP-rhMPV可在低熔点琼脂糖凝胶覆盖层下形成分界较为清晰的绿色荧光集落,接种后3 d荧光空斑相对独立,便于计数.蓝斑形成法在感染后第5天蓝斑较大,易观察.此前拯救获得的GFP-rhMPV在宿主细胞Vero-E6中的复制滴度可达1×106 PFU以上.结论 成功建立了GFP-rhMPV的空斑形成实验滴度定量检测方法,为hMPV的致病机制、防治手段研究奠定了基础.     

Replication and plaque formation of swine hemagglutinating encephalomyelitis virus (67N) in swine cell line, SK-K culture

Swine hemagglutinating encephalomyelitis virus (HEV), 67N strain, adapted to suckling mouse brain, grew readily in a porcine cell line, SK-K cell culture with cytopathic effect (CPE) consisting of syncytium formation and detachment of fused cells and round cells from glass surface. After further passages in SK-K cell monolayers with undiluted culture fluid, CPE developed earlier and became complete within 48 h postinoculation (p.i.). Viral specific antigen was detected in the cytoplasm of the infected SK-K cells by indirect immunofluorescence using rabbit antiserum against the mouse-passaged virus. The SK-K-passaged virus as well as the original mouse-passaged virus formed clear plaques on SK-K cell monolayers under simple overlay medium. The plaque assay system for HEV 67N was established by studying various factors influencing the plaque formation in the SK-K cell cultures. By this system more than 10(6) PFU/0.2 ml of the virus yield was detected in the fluid phase of the infected cultures at 48 h p.i. The SK-K-passaged virus caused fatal infection in 4-week-old mice by intracerebral inoculation, but was inhibited by rabbit antiserum against the mouse-passaged virus. Plaque formation and hemagglutinating activity of the virus were specifically inhibited by antisera against the mouse-passaged and SK-K-passaged 67N virus.     

A plaque assay for feline panleukopenia virus

Plaque formation with representative strains of feline panleukopenia virus (FPV) has been obtained using a permanent line of feline kidney cells under agarose overlay. FPV-infected cells appear as white plaques after neutral red staining. Plaque size is determined by the extent of cell division in the infected monolayer. FPV assay by the plaque procedure is rapid and gives infectivity titres which exceed those determined by the common inclusion body and immunofluorescent assays of FPV by a factor of about 100 and 10, respectively. Moreover, the plaque assay offers an effective means for the quantification of neutralizing antibodies in feline sera as well as for the detection of heat-stable substances in bovine sera which strongly interfere with replication of the virus.     

Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses

A novel method was developed for the precise quantitation of viruses using infrared fluorescent detection of foci of infection in conventional cell culture plates. In this assay, termed the infrared fluorescent immunofocus assay (IR-FIFA), appropriate cell cultures were infected with serial dilutions of hepatitis A virus (HAV) or measles virus (MV) and maintained with a semi-solid overlay for 1-5 days. Cell monolayers were fixed with formaldehyde, and then stained in succession with a primary monoclonal antibody and an Alexa Fluor 680 conjugate. Foci of infection (analogous to plaques) were detected by scanning culture plates using the Odyssey infrared imaging system and counted to determine the virus titre, expressed as focus forming units (FFU) per mL, as is done for conventional plaque assays. HAV and MV were used as models of minimally cytopathic viruses, and showed a linear dose-response between focus formation and virus dilution. Viral titres calculated using this method were comparable to conventionally used methods. The IR-FIFA was also successfully adapted to quantify duck hepatitis B virus (DHBV) as a model for a non-cytopathic virus. This simple and sensitive assay will have wide use for the quantitation of non-cytopathic and minimally cytopathic viruses.     

A novel plaque assay system for paramyxoviruses

A combination of 5% allantoic fluid and 200 micrograms/ml DEAE-dextran with 30 mM MgCl2 used as a supplement to normal overlay medium was found to give large, clear plaques on monolayers of secondary chick embryo fibroblasts infected with avirulent strains of Newcastle disease virus which did not produce plaques without these additions. This modified overlay also allowed plaque assay of Sendai virus strain Z, and avoided any requirement for special cell lines or the use of serum-free medium.