Plaque Assay for Pneumonia Virus of Mice

A plaque assay for the quantitation of pneumonia virus of mice is described. To obtain reproducible plaque formation, proteolytic enzymes had to be incorporated in the overlay medium. The plaque morphology observed in the presence of pancreatin and chymotrypsin was superior to that seen with trypsin. Although the plaque assay was found to be slightly less sensitive than the TCID(50) determination described by Harter and Choppin, it enables cloning of the virus by plaque selection and permits further study of pneumonia virus of mice.     

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.     

Role of the host cell in persistent viral infection: Coevolution of L cells and reovirus during persistent infection

Mutant L cells, designated LR cells, were isolated after “curing” a persistently infected cell line (L/C) with antireovirus serum. The LR cells were shown to be virus-free; no reovirus was detectable by infectious center assays, plaque assays, presence of viral proteins, presence of viral dsRNA and immunofluorescence studies. Persistent infections were readily established in LR cells following infection with either cloned, low passage wild-type reovirus or cloned, low passage reovirus isolated from carrier cultures. Reovirus isolated from carrier cultures, however, grew much better than wild-type reovirus in LR cells and showed complete dominance over wild-type reovirus in coinfection experiments. Infection of LR cells with wild-type reovirus resulted in a low-level persistent infection with inefficient viral replication; these mutant L cells were partially resistant to infection with wild-type reovirus. In contrast, infection of the mutant L cells with virus isolated from the persistently infected cells resulted in a persistent infection accompanied with efficient viral replication. Infection of the original L cells with either wild-type reovirus or reovirus isolated from the persistently infected cells resulted in a lytic infection with no surviving cells. Thus the host cell plays a crucial role in the maintenance of persistent reovirus infection. Our results show that there is a coevolution of both mutant L cells and mutant reovirus during persistent infection.     

Application of a serum-free medium for the growth of Vero cells and the production of reovirus

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

Multiplicity reactivation of reovirus particles after exposure to ultraviolet light

McClain, Mary E. (California State Department of Public Health, Berkeley), and Rex S. Spendlove. Multiplicity reactivation of reovirus particles after exposure to ultraviolet light. J. Bacteriol. 92:1422-1429. 1966.-Exposure of reovirus suspensions to moderate doses of ultraviolet light results in essentially exponential inactivation of infectivity to survivals of 10(-2) to 10(-3). With suspensions of sufficiently high particle concentration, larger doses of ultraviolet light (6 to 12 min) are associated with multiplicity reactivation (MR) which is demonstrable both by immunofluorescent-cell count and by plaque assay in FL human amnion cells. Similar effects are produced by photodynamic inactivation in the presence of proflavine, but not by thermal inactivation at 50 C. All three reovirus types exhibit MR under appropriate conditions, and all three interact in mixed ultraviolet suspensions with high efficiency. Progeny from FL cells infected under conditions of MR were as infectious as those of unirradiated inocula, with yields per cell ranging from 10(4) to 4 x 10(4) infective units.     

Mouse Hepatitis Virus (MHV-2)

Various factor sinfluencing the plaque formation of mouse hepatitis virus (MHV-2) in DBT cell monolayers were studied and a practical method for plaque assay was developed. Infected DBT cells yielded high-titered virus and were a satisfactory source of complement-fixing viral antigen. The predominant cytopathic effect of MHV-2 in DBT cells was cell rounding and detachment, but no syncytial formation was observed. Fluorescent antibody staining revealed specific fluorescence only in the cytoplasm of infected DBT cells. In one-step growth experiment, newly formed virus was first recognized within 4-hr postinfection and showed subsequently a rapid exponential increase. Release of newly formed virus from the cell was rapid, and a continuous release lasted for a certain period of time. The average per-cell yield of active virus was estimated to be about 6–7 × 10² plaque-forming units.     

Mouse hepatitis virus (MHV-2). Plaque assay and propagation in mouse cell line DBT cells.

Various factor sinfluencing the plaque formation of mouse hepatitis virus (MHV-2) in DBT cell monolayers were studied and a practical method for plaque assay was developed. Infected DBT cells yielded high-titered virus and were a satisfactory source of complement-fixing viral antigen. The predominant cytopathic effect of MHV-2 in DBT cells was cell rounding and detachment, but no syncytial formation was observed. Fluorescent antibody staining revealed specific fluorescence only in the cytoplasm of infected DBT cells. In one-step growth experiment, newly formed virus was first recognized within 4-hr postinfection and showed subsequently a rapid exponential increase. Release of newly formed virus from the cell was rapid, and a continuous release lasted for a certain period of time. The average per-cell yield of active virus was estimated to be about 6-7 X 10(2) plaque-forming units.     

Comparison of Microtiter Procedures with the Plaque Technique for Assay of Vesicular Stomatitis Virus

This paper describes a microprocedure for the tissue culture assay of vesicular stomatitis virus (VSV) and compares the sensitivity of the method with the conventional plaque assay of viral concentration. Microtiter and plaque assay methods were used in titrations, neutralization tests, and thermoinactivation studies with this virus in chick embryo fibroblasts (CEF) and L, HeLa, and PK cell lines. Titration experiments with VSV by the microtiter procedure on preformed monolayers were significantly more sensitive than the plaque assay (P = 0.05). The results of the neutralization tests and thermoinactivation studies also showed greater ability to detect residual virus by the microtiter procedure (P = 0.10). In addition, the microtiter procedure was simpler, less costly, and more rapid than the plaque assay.     

Design and characterization of a compact dual channel virus counter.

BACKGROUND: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard." Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. METHODS: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels. RESULTS: The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay. CONCLUSIONS: The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (approximately 100 microl).     

Dengue Virus Plaque Development in Simian Cell Systems: II. Agar Variables and Effect of Chemical Additives

Dengue type 2 virus, strain New Guinea B, plaqued with equal facility and titer under overlays containing six different grades of commercial agar in the LLC-MK₂ cell system. Doubling the agar volume on LLC-MK₂ cell monolayers increased the plaque development time of dengue type 1, strain Hawaii. Storage of agar at 56 C reduced or totally abolished dengue type 4, strain H-241, plaque titer in LLC-MK₂ cells. The influence of six known virus plaque-enhancing compounds on plaque development of all dengue virus serotypes was studied in two continuous simian kidney cell lines, LLC-MK₂ and Vero. In the absence of any chemical additive, plaque development of all dengue serotypes was more rapid (4 to 10 days) in the LLC-MK₂ line than in the Vero line (6 to 13 days). Increased plaque development time of type 1, strain Hawaii, by pancreatin and plaque-size doubling of dengue types 1 and 4 was the only advantage conferred by the addition of six chemical additives in the LLC-MK₂ cell system. Dengue types 1 and 6 failed to plaque in the Vero cell system unless aided by a plaque-enhancing compound; plaques of dengue types 2, 3, 4, and 5 appeared sooner (2 days) and were increased in plaque diameter. The optimal DEAE concentration for plaquing dengue type 1, strain Hawaii, was 100 μg/ml; plaque development either failed at lower concentrations or was inhibited at higher (200 μg/ml) concentrations.     

Adaptation of Plaque Assay Methods to the in Vitro Quantitation of the Radiation Leukemia Virus

A modification of the XC cell procedure for murine leukemia virus assay which yields quantitative data over a wide range of virus concentrations is described. By using serial passage of infected cell cultures and reversal of the plating sequence in the XC procedure, titers of radiation leukemia virus (RadLV) were obtained which were about 10-fold higher than those found by using the conventional assay. By using the modified procedure, it was observed that, even at high multiplicities of infection, less than 10% of the cells function as infective centers, although the proportion increases with serial passage. It was also observed that exposure of infected cells to UV light, which is commonly used to make plaques more visible in the conventional XC cell test, inhibits plaque formation in the RadLV system. Substitution of X irradiation for UV exposure improved plaque visibility without loss of sensitivity.     

Reovirus Growth in Cell Culture Does Not Require the Full Complement of Viral Proteins: Identification of a ς1s-Null Mutant

The reovirus ς1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis. Although the function of ς1s is not known, the ς1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express ς1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the ς1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain ς1s. To determine whether ς1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells. After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether ς1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein ς1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.     

Proteins of Polyhedral Cytoplasmic Deoxyvirus II. Nucleotide Phosphohydrolase Activity Associated with Frog Virus 3

A nucleotide phosphohydrolase is firmly associated with a purified polyhedral cytoplasmic deoxyvirus, frog virus 3. This adenosine triphosphatase is distinguishable from known mammalian cell adenosine triphosphatases and from adenosine triphosphatase of an unrelated cytoplasmic replicating virus grown in the same host cell. The enzyme activity has a high specificity for adenosine triphosphate; the product of the reaction is adenosine diphosphate. The presence of similar activities in reovirus and poxvirus indicates that adenosine triphosphatase might have a function in the replication of these viruses.     

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.     

Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples

The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.     

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.     

Protamine precipitation of two reovirus particle types from polluted waters.

Two forms of virus particle are released from reovirus-infected cell cultures, infectious reovirus and potentially infectious reovirus (PIV). PIV particle forms have a complete outer coat and are not infectious until the outer coat is altered or removed. The PIV concentration in polluted waters, however, has not been determined. Protamine sulfate precipitation, using 0.25% fetal bovine serum and 0.005% protamine sulfate for the first precipitation of the sample and 0.0025% for the second, was employed to concentrate infectious reovirus and PIV from water and sewage. Infectious reovirus and PIV particles were concentrated over 500-fold from river water inoculated with virus, and virus recoveries of between 80 and 100% were achieved. Virus precipitates stored at -20 degrees C as a protamine-virus concentrate showed a 5% loss of PIV after 14 days. Virus preparations were assayed, before and after treatment, with 200 micrograms of chymotrypsin per ml, using a fluorescent-antibody procedure. Protamine sulfate precipitation and fluorescent-antibody detection are effective ways to recover and assay reoviruses present in raw sewage.     

The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein.

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.