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.     

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.     

Flow cytometry-based assay for titrating dengue virus

Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.     

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.     

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.     

The isolation and characterization of the MP and FP plaque variants of Galleria mellonella nuclear polyhedrosis virus

Two plaque morphologies were detected in assays of Galleria mellonella nuclear polyhedrosis virus preparations which had been serially passaged in the TN-368 cell line. The FP (few polyhedra) plaques were larger (0.7-mm average diameter) and the infected cells contained less than 10 polyhedra each. The MP (many polyhedra) plaques were smaller (0.5-mm average diameter), and the infected cells contained greater than 10 each. Only the FP variant could be purified by plaque picking and remained pure through 10 serial injection passages in last-instar Galleria mellonella larvae, while the MP variant always produced FP plaques after serial passage in cell cultures or by injection of larvae. Bioassays of MP and FP polyhedra demonstrated a 350-fold reduction in virulence for FP polyhedra. Electron micrographs of these polyhedra showed that both variants were MNPV type viruses, although the FP polyhedra were predominantly devoid of nucleocapsids. Electron microscopic observations of MP- and FP-infected cell cultures revealed an apparent deficiency in de novo membrane synthesis in FP infections. SDS-polyacrylamide gel electrophoresis of cell-released nonoccluded virus proteins demonstrated several differences in protein composition between the two variants. The evidence indicates that the FP variant of G. mellonella nuclear polyhedrosis virus is a stable genotypic variant which arises from spontaneous mutation of the MP variant.     

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.     

Susceptibilities of 14 cell lines to bluetongue virus infection.

The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations.     

Effect macrophage activation on infection with Treponema pallidum.

An in vitro method is described to measure the inhibitory activity of murine peritoneal exudate cells against viral plaque formation by a bovine herpes-virus-infectious bovin'e rhinotracheitis virus. Microtiter plates containing 96 bovine kidney cell monolayers were infected with a range of virus concentration and peritoneal exudate cells were subsequently added. When a sufficient number of cells was added, viral plaques were not detectable and free infectious virus did not occur in the culture fluids. The inhibitory cell type adhered to glass and was presumably a macrohage. Although inhibitory of viral plaques was presumably a macrophage. Although inhibition of viral plaques was complete and free virus could not be detected, virus was not eliminated from the monolayers since on removal of cells, the degree of virus cytopathology and yield of virus after a further 48 h of incubation was the same as in 48-h infected control monolayers. The significance of peritoneal exudate-cells-induced virus suppression as a model to understand herpesvirus latency is briefly discussed.?Author     

Rapid titration of bovine, caprine and human RS virus by a micro-immunoperoxidase assay using a monoclonal antibody and a permissive ovine kidney cell line

An indirect immunoperoxidase micro-assay, using a continuous cell line derived from ovine kidney cells (OK) and a previously characterized monoclonal antibody (7C2), specific for an exposed and highly conserved epitope of the fusion protein of different strains of RS virus, was used advantageously to rapidly titrate bovine, caprine and human strains of RSV by either quantal (TCID50) or plaque forming assays. Virus titers, obtained in less than 36 h, were in agreement with those obtained by the conventional plaque assays which required an incubation period of 4 days or more. This assay is also applicable to micro-neutralization of fusion inhibition assays for testing serum or screening monoclonal antibodies.     

A feline kidney cell line-based plaque assay for feline calicivirus,a surrogate for Norwalk virus

Feline calicivirus (FCV) has been used by researchers as a surrogate for Norwalk virus (NV), since they share a similar genomic organization, physicochemical characteristics, and are grouped in the same family, Caliciviridae. Unlike NV, however, FCV can grow in established cell lines and produce a syncytial form of cytopathic effect. In this report, we describe the development and standardization of a plaque assay for FCV using monolayers of an established line of feline kidney (CrFK) cells in 12-well cell culture plates. The assay method has demonstrated reproducibility, ease of performance and resulted in clear plaque zones, readable in 24 h after virus inoculation. The infectivity titre of the virus by this plaque assay agreed well with tissue culture infectious dose(50) (TCID(50)) determinations. The described plaque assay would be a valuable tool in conducting various quantitative investigations using FCV as a model for NV and Norwalk-like viruses (NLV).     

Development of plaque assays for adenoviruses 40 and 41

Enteric adenoviruses, important agents of infantile gastroenteritis, are difficult to culture with low titers and limited CPE. Consequently, few plaque assays have been reported and none are used routinely by investigators who may need reproducible quantitative assays for these viruses. CPE in A549 cells (an epithelial lung carcinoma cell line) was induced by isolates of human adenovirus (HAdV) serotypes 40 or 41 that were obtained by prior limited passage in primary cynmolgous monkey kidney (pCMK), human embryonic kidney (HEK), and Graham 293 cells. CPE with HAdV 40 (Dugan strain) and HAdV 41 (Tak strain) inoculated in A549 cells was also observed. Monolayers of A549 cells were inoculated with a low multiplicity of infection (MOI) of the archived stock isolates and harvested at days 10-14 with full CPE. Subsequent passages were harvested in as few as 7 days with 100% CPE to prepare virus stocks for plaque assay. Large individual plaques under agarose overlay were picked prior to staining and clonal stocks prepared. Titers of final stock preparations after six to eight passages in A549 cells were in the range of 5 x 10(7)-1 x 10(8)PFU/ml, which provides adequate virus for quantitative recovery studies. The particle to infectivity (P:I) ratios of the early passages of virus stocks were in the range reported previously. The ratio of non-infectious to infectious particles decreased with successive passages of HAdVs 40 and 41 in A549 cells. The specificity of the assay was confirmed by neutralization of plaques with type-specific antisera. Furthermore, sequence analysis of the HAdVs 40 and 41 plaque forming stocks ruled out contamination with any other HAdVs. The plaque assay developed will be useful for evaluation of virus recovery methods from water, food or other environmental matrices, as well as determination of the efficacy of water treatment techniques for inactivation of these viruses.     

Isolation of large and small plaque variants of theAutographa California nuclear polyhedrosis virus

Summary Variants of theAutographa californica nuclear polyhedrosis virus which produce large (1.4 mm) or small (0.5 mm) plaques onSpodoptera littoralis cells have been isolated. Yields of extracellular virus and polyhedra by the large plaque variant were six-fold higher and 140-fold lower, respectively, than those obtained with the small plaque variant. However the two variants could not be distinguished whenSpodoptera frugiperda cells were used.With 1 Figure     

TCID50 determination by an immuno dot blot assay as exemplified in a study of storage conditions of infectious pancreatic necrosis virus.

Some cell lines are difficult to grow in confluent monolayers and, therefore, the plaque assay cannot be applied since plaques may be hard to distinguish from other blank areas of the cell monolayers. To avoid this problem a rapid and sensitive immuno dot blot TCID50 method was developed using antibodies against virus antigens to detect infection and virus production. An alternative statistical method was developed to treat the scoring data and thereby obtained a coefficient of variation of 10%. To speed up the total procedure and to increase the proliferation rate of cells grown in 96-well cell culture plates, the plates were pretreated for 4 h at 4 degrees C with growth medium obtained from cell culturing flasks containing confluent cell monolayers. This immuno dot blot TCID50 method was applied for a study of the infectivity maintenance upon storage of infectious pancreatic necrosis virus (IPNV). Storage of IPNV at -70 degrees C with a cryoprotective agent (10% glycerol) preserved the TCID50 level even after as many as ten cycles of freezings and thawings, whereas the infectivity decreased by four orders of magnitude after storage at 4-8 degrees C for 2 months in the salt buffer used commonly.     

A colorimetric assay for viral agents that produce cytopathic effects

Many animal viruses produce cytopathic effects in their host cells during a productive infection. While some virus infections can be assayed by the production of plaques, many viruses, while producing cytotoxicity, do not easily form plaques, or do not form plaques at all. Additionally, viruses within families such as the parvoviruses may have different preferred forms of titration making comparative virology difficult even among related groups. Porcine parvovirus (PPV), canine parvovirus (CPV), and minute virus of mice (MVM) are usually titrated using different infectivity assays. A direct comparison of infectious virus titer between these parvoviruses was sought, and a tetrazolium salt assay, MTT has been applied to measure cytopathic effect produced by viral infection for different members of the parvovirus family. Infectious PPV measured using the MTT and the TCID50 assays exhibited excellent correlation and titers for CPV and MVM were consistently duplicated using the MTT assay. The MTT assay was also applied to an unrelated virus, Sindbis, which is routinely titrated by plaque assay. MTT titration of Sindbis virus mutants was found to be valuable for preliminary screening. This assay can be adapted, by correlation to an accepted titration method, to any viral system which produces measurable cytopathic effect.     

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.     

Rapid, quantitative, semiautomated assay for virus-induced and immune human interferons.

An improved human interferon (IF) assay is described. This procedure is based on the ability of encephalomyocarditis virus to replicate in WISH cell microcultures with the production of discrete plaques in the presence of a liquid tissue culture medium. Performance of 50% plaque reduction endpoint assays in micro-culture required only 0.1 ml of specimen for determinations using duplicate dilutions beginning at 1:3. Semiautomated equipment facilitated simultaneous in situ dilution and distribution of multiple IF samples in cultures containing preformed WISH cell monolayers. An incubation period of 5 to 6 h was adequate for development of maximal antiviral activity by both virus- and immune-induced IF. Sensitivity of the encephalomyocarditis microplaque reduction assay was comparable to that of other commonly used techniques. The method is rapid, can be completed within 30 h from the beginning of the IF assay, and is able to accommodate as many as 40 to 50 samples at a single time. Encephalomyocarditis microplaque reduction is suitable for the quantitation of IF as an antiviral agent or a lymphokine.     

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.     

Quantitation of flaviviruses by fluorescent focus assay

An indirect immunofluorescence assay for quantitation of flaviviruses was developed as an alternative to the standard plaque assay. The assay was validated with West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Dengue virus (DENV) types 1-4. Vero cells were plated in 8-well chamber slides, and infected with 10-fold serial dilutions of virus. About 1-3 days after infection, cells were fixed, incubated with specific monoclonal antibody, and stained with a secondary antibody labeled with a fluorescent tag. Fluorescent foci of infection were observed and counted using a fluorescence microscope, and viral titers were calculated as fluorescent focus units (FFU) per ml. The optimal time for performing the fluorescent focus assay (FFA) on Vero cells was 24 h for WNV, and 48 h for SLEV and the four DENV serotypes. In contrast, the time required to complete a standard Vero cell plaque assay for these viruses range from 3 days for WNV to 11 days for DENV-1. Thus, the FFA method of virus titration is useful for viruses whose plaques develop slowly. In addition, these viruses can be quantitated by FFA on a mosquito cell line (C6/36), which does not support plaque formation. The FFA for flaviviruses was validated for accuracy, precision, specificity, and robustness of the assay.