Relationship of Plaque Size and Virulence for Chickens of 14 Representative Newcastle Disease Virus Strains

Ability of 14 Newcastle disease virus strains to produce large plaques was related to virulence for chickens. Plaque-size comparisons were made under standard conditions in chick embryo cell monolayers. All plaque-producing strains showed a range of plaque sizes modified to a degree by the overlay medium used. An increase in size was found for most strains under methyl-cellulose overlay medium. Markedly larger plaques were found under this medium for both Calif-RO and Calif-CG strains. Heterogeneity in plaque size was most pronounced in velogenic (high virulence) strains. Only populations of small plaques were found in mesogenic (intermediate virulence) strains, and plaques were rarely found in lentogenic (low virulence) strains. Statistical analysis showed that the plaque size of velogenic strains differed significantly from mesogenic strains. None of the 11 plaque-producing strains had a normal distribution of plaque sizes, owing primarily to the presence of different genotypes within the plaquing population of a strain. This was demonstrated by derivation of clones from two of the strains. The populations of the large (Herts L) and small (Herts S) clear plaque clones derived from Eng-Herts were homogenous and distinct from one another on the basis of plaque size. Herts L was more virulent than Herts S. Although Herts L became more heterogenous in respect to plaque size upon repeated passage in embryonated eggs, no decrease in virulence of the strain was observed.     

Plaque Formation by Mumps Virus and Inhibition by Antiserum

Boston and ABC strains of mumps virus produced plaques approximately 1.0 mm in diameter in monolayers of BGM cells. The plaques were circular and either clear or target-like in form. Ricki strain virus produced plaques of similar size and form but, in addition, a red plaque was observed with this agent. The vaccine strain of mumps virus, Jeryl Lynn, produced minute clear plaques approximately 0.3 mm in diameter. Incorporation of diethylaminoethyl (DEAE)-dextran into the overlay medium did not affect the size difference between Jeryl Lynn plaques and those of the other strains. However plaques of the Jeryl Lynn and Ricki strains were more easily visualized when the overlay medium contained 400 mug/ml of DEAE-dextran. Simultaneous titration by plaque formation and roller tube infectivity showed that these two methods were of equal sensitivity. Virus neutralization by antibody was demonstrated by plaque reduction. Rise in antibody titer was observed in sera from human and animal infection, human vaccination, and rabbit immunization.     

Evaluation of a Plaque Assay for the Maedi-Progressive Pneumonia-Visna Viruses

A simple and direct plaque assay for maedi virus, two strains of progressive pneumonia virus, and two strains of visna virus has been developed and evaluated. The technique allows the plaques formed by these viruses to be localized without disturbing the host-cell substrate of sheep choroid plexus cells or the gelled maintenance medium over the host-cell monolayer. Diethylaminoethyl-dextran supplementation of the medium used to overlay strain K796 visna virus-infected cultures decreases the time required for maximum plaque development from 12 to 10 days, enhances the contrast of the plaques, increases the titer of plaque-forming units, and permits a plaque size heterogeneity to be realized. Both large and small plaques occur in cultures infected with the visna viruses, one strain of progressive pneumonia virus, or maedi virus. In contrast, the plaques observed in cultures infected with the second strain of progressive pneumonia virus are relatively homogeneous in size.     

Enhanced Detection of Western Equine Encephalitis Virus Plaque Variants in Crowded Cultures and Plaque Progeny—Potential Use in Genetic Studies

Enhanced detection of large-plaque variants of Western equine encephalitis virus in the presence of large numbers of small-plaque Western encephalitis virus was achieved by using higher bicarbonate concentrations in the agar-overlay medium and increasing concentrations of CO(2) in an enclosed environment during the time plaques were developed with neutral red. By this technique we were able to detect large-plaque mutants in cloned small-plaque suspensions for the first time and to show an increase in detectable plaque progeny in plaques developed by neutral red. Further increase in the number of progeny per plaque was determined by assaying extracts of agar overlaying cultures containing known numbers of plaques. The significance of these findings for potential use in mutational studies is discussed.     

Separation of Plaque-type Variants of Encephalomyocarditis Virus by Chromatography on Calcium Phosphate

Encephalomyocarditis (EMC) virus (K-2 strain) was separated into two distinct peaks of optical density by chromatography on calcium phosphate, eluting at 0.17 and 0.31 m, respectively. The virus in these peaks could not be distinguished by infectivity (plaque-forming units), hemagglutinin activity, electron microscopic appearance, sedimentation coefficient, or base ratios. However, variants producing large plaques predominated in the first peak eluted, whereas variants producing small plaques were concentrated in the second peak. When plaque-purified large and small plaque variants were chromatographed individually, each gave a single peak eluting at 0.16 and at 0.43 m, respectively. However, one small plaque type isolated was eluted in the 0.17 m region expected for large plaque variants, and not with the bulk of the small plaque variants. There were indications that EMC virus (K-2 strain) could be separated into more than two peaks by use of selected elution gradients. The possibility that there may be a relationship between plaque size and point of elution from calcium phosphate is discussed.     

Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

检测流行性出血热病毒滴度和中和抗体效价的半微量空斑法

建立了检测流行性出血热病毒滴度和中和抗体效价的半微量空斑法。小牛血清与胎牛血清的培养效果无差异。7株不同来源的出血热病毒均能在E6细胞上形成空斑。接种的病毒浓度与形成的空斑数呈直线关系。用空斑法测得的病毒滴度稍低于荧光TCIE50滴定法。空斑减少中和试验的敏感性较荧光中和试验高30倍左右。同时还初步表明了本方法可用于流行性出血热病毒的抗原性分析。     

Plaque Formation in Chick Embryo Fibroblast Cells by Chlamydia Isolated From Avian and Mammalian Sources

Fifteen strains of chlamydial agents, 2 Chlamydia trachomatis and 13 Chlamydia psittaci, were tested for plaque formation on chick embryo fibroblast cells. C. trachomatis strains did not form plaques; 12 strains of C. psittaci did form plaques and 1 strain did not. The distribution of plaque sizes with C. psittaci strains was studied and it was found that the 12 strains could be tentatively grouped into three plaquing types: small (<0.50 mm), intermediate (0.50 to 0.90 mm), and large (>0.90 mm). Small plaques were predominantly associated with strains M56, ET, CP3, CT4, EBA, CS1, and CT2. Intermediate size plaques were predominantly associated with strains NJ1, VT1, and H81. Large plaques were predominantly associated with strains WC and OG. Plaque size appears to be related to virulence of Chlamydia for laboratory animals.     

A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.     

Differentiation of Temperature Variants of Influenza A2 Viruses on the Basis of Plaque Formation

The ability of temperature variants of influenza A2 virus to produce plaques in chick embryo kidney tissue culture was studied at different temperatures. Definite differences in efficiency of plaque formation by cryophilic and thermophilic strains were observed at low and high temperatures. Differentiation of the temperature variants appears to reside in a number of genetic markers, designated rct-40, rct-28, and plaque size (S). Virulence of influenza A2 virus, enhanced after prolonged cultivation at high temperature, is probably related to its greater efficiency of plating at 40 C (rct-40(+)), formation of larger plaques at optimal (36 C) and high (40 C) temperatures, and to loss of ability to form plaques at 28 C (rct-28(-)).     

Comparative Studies on Large- and Small-Plaque-Forming Clones of Newcastle Disease Virus (Miyadera Strain)

The Miyadera strain of Newcastle disease virus (NDV) consisted predominantly of virus particles forming small plaques on monolayers of chick embryo fibroblasts (CEF), and contained small amounts of virus particles forming large plaques. These large- and small-plaque-forming clones of this virus (NDV-L and NDV-S) were isolated. The small size of the NDV-S plaques did not appear to be due to an agar inhibitor. NDV-L produced a much higher yield of infective virus particles in CEF and they were released more completely from the infected cells than were those produced by NDV-S. The yield of infective virus of NDV-L per cell from cultures of CEF was comparable to the yield from the allantoic cells. The infectivity/hemagglutinin ratio for NDV-L from CEF was as high as the ratio for virus from the allantoic cells, but the ratio for NDV-S from CEF was lower. NDV-S demonstrated an autointerference phenomenon in CEF when infected at high multiplicities, but NDV-L did not. Contrary to virus multiplication, NDV-S exhibited a more rapid and marked cytopathic effect on monolayers of CEF than NDV-L. In the allantoic cavity of eggs NDV-S produced slightly higher virus yields than NDV-L. No correlation existed between plaque size of the two viruses and the capacity to induce interferon synthesis or the susceptibility to the action of interferon. The properties of both distinctive plaque isolates were stable on egg passage.     

Markers of Rubella Virus Strains in RK13 Cell Culture

When tested on RK(13) cell cultures, strains of rubella virus could be differentiated by their ability to form small or large plaques. Large plaques were produced by the HPV-77 and Cendehill strains, and also by a laboratory stock strain (West Point), after only 14 passages in RK(13) culture. Five wild-type rubella viruses, isolated and passaged only a few times in African green monkey kidney tissue culture, grew well in RK(13) cell culture, but they were sensitive to agar inhibitors and, therefore, formed small plaques. On the other hand, RA27/3, an attenuated strain grown in WI-38 human fibroblast cells, developed low titers in RK(13) cells and also produced small plaques. We concluded that the morphological differences between small-plaque and large-plaque viruses depended on their sensitivity to agar inhibitors and on the pH of the medium during plaque formation.     

A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line

This is the first report of plaque formation by a pathogenic insect virus. Trichoplusia ni (TN-368) cells overlaid with medium containing 0.6% methyl cellulose continued to multiply, developed into monolayers, and produced plaques after infection with alfalfa looper nuclear polyhedrosis virus. Viral polyhedral inclusion bodies were first observed 24 hr after exposure of cells to virus, and plaques continued to increase in size for 72 hr. Two different types of plaques were observed: one in which all cells had many polyhedra in their nuclei, and another in which few cells had inclusion bodies. When virus from either plaque was injected into T. ni larvae, they died of typical nuclear polyhedrosis virus disease. The assay was reproducible, and plaque numbers were related to virus concentration.     

Properties of Venezuelan Equine Encephalomyelitis Virus Grown In Vivo

One intracerebral passage of either the parent egg seed (PES) or an attenuated variant (10t) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus in young adult mice produced progeny that were no longer differentiated unequivocally on the basis of plaque size. Plaques averaging about 2 mm in diameter, which was somewhat smaller than those formed by the PES virus and larger than those of the 10t strain, were formed by both strains. Seven serial passages of the PES virus in mouse brain failed to alter its virulence appreciably. In contrast, passage in mouse brain progressively changed the properties of the attenuated 10t strain. A substrain was isolated that possessed virulence similar to that of the PES virus and formed small plaques similar to those of the 10t strain. These findings showed a unique dissociation between the plaque size and virulence of the 10t strain. The new substrain differed from the PES virus and the 10t strain in its capacity for growth in mouse tissues after intraperitoneal inoculation. The substrain multiplied poorly in splenic tissue, which supports growth of the PES and 10t strains, but grew to high titers in the brain, which does not support appreciable growth of the 10t strain.     

Enhancement of St. Louis Arbovirus Plaque Formation by Neutral Red

Experiments with St. Louis encephalitis (SLE) virus have shown that neutral red enhances the plaque size in duck embryo cell cultures. This may represent a new method for genetic studies of SLE virus population. In a mosquito pool specimen NR(+) and NR(-) particles were demonstrated. By intracerebral passage in mice, there is a selection of NR(+) particles. Similar effects were not shown for eastern, western, and California encephalitis viruses. Plaques formed by the latter virus, however, were significantly reduced in number by neutral red.     

Gamma Ray-Induced Small Plaque Mutants of Western Equine Encephalitis Virus

Small plaque mutants of Western equine encephalitis virus were obtained from the surviving fractions of wild-type virus which was irradiated with gamma rays. The frequency with which small plaque mutants appeared in the surviving fraction increased with the radiation dose. These mutants were not more resistant to radiation than wild-type virus. The growth rate of a mutant, S127, was lower than that of wild-type. Clonally purified mutant virions presented two peaks in a velocity sedimentation profile; peak 1 corresponded to the peak of wild type and peak 2 moved faster than peak 1. Virions of both peaks were infectious and consistently formed small plaques in chicken embryo cells. Virions reisolated from either peak and grown in chicken embryo cells also revealed two peaks in sedimentation analysis. In the electron microscope examination peak 2 proved to consist of giant form particles, each of which contained more than one nucleoid surrounded with a common envelope. Despite this remarkable morphological difference, densities of the wild-type and S127 mutant virions were similar in cesium chloride gradients. The RNAs and proteins of mutant virions could not be distinguished from those of wild type on the basis of size or charge.     

Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR)

Visualization of bacteriophage plaques may be enhanced by addition of ferric ammonium citrate and sodium thiosulfate (FACST) or 2,3,5-triphenyltetrazolium chloride (tetrazolium red, TZR) to the soft agar layer of a traditional bacteriophage plaque assay. Background color from these reagents improved contrast between clear plaques and turbid host lawns in trypticase soy agar (TSA) plates. Enhancement by FACST is based on reaction with hydrogen sulfide gas (H2S) produced by some strains of bacteria and was tested here using H2S+ and H2S- strains of Salmonella enterica subsp. enterica with a bacteriophage (Podoviridae) isolated from swine lagoon effluent. Only the H2S+ strain produced dark brown-black color in FACST-amended agar. Both strains showed bright pinkish-red color in TZR-amended agar. Color intensity for both reagents decreased with decreasing concentrations of the reagents. Contrast in FACST-amended plates appeared greater than that with TZR, but diminished after 12 h, while contrast in TZR-amended plates remained constant. At the concentrations tested, neither reagent affected plaque counts in the H2S+ strain. The FACST should be useful in bacteriophage plaque assays with H2S+ strains of Salmonella and other H2S+ bacteria.     

A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.     

An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages

This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis . Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage–host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.