Selection of Temperature-Sensitive Mutants During Persistent Infection: Role in Maintenance of Persistent Newcastle Disease Virus Infections of L Cells

Virus mutants (NDV(pi)) recovered from L cells persistently infected with Newcastle disease virus (NDV, Herts strain) are temperature-sensitive (ts) at 43 C, although the wild-type virus (NDV(o)) which initiated the persistent infection replicates normally at that temperature. To study the relationship between the ts marker of NDV(pi) and the other properties which distinguish this virus from NDV(o), NDV(pi) ts(+) revertants were selected at the nonpermissive temperature and NDV(o) ts mutants were generated by treating NDV(o) with nitrous acid. Spontaneously-occurring ts mutants in the Herts NDV population were also isolated. The different virus populations were characterized with regard to plaque size, virulence for eggs, and thermal stability of infectivity, hemagglutinin, and neuraminidase. The NDV(pi) ts(+) revertants, although no longer temperature-sensitive, retained NDV(pi) properties, whereas both spontaneously-occurring and mutagen-induced ts mutants remained wild-type in their other properties. These findings showed that the properties which characterized NDV(pi) were independent of the ts marker. However, the ts marker and the other markers of NDV(pi) were coselected during the persistent infection, and the combination of those markers appeared to be important in the outcome of NDV infection of L cells. NDV(pi) replicated productively in L cells, whereas NDV(o), the NDV(pi) ts(+) revertants, and the spontaneously-occurring ts mutants all yielded covert infections in L cells. The role of the selection of ts mutants in persistent infection was confirmed as follows: L cells were persistently infected with NDV(pi) ts(+) revertants and NDV(o) ts mutants. Virus recovered from the persistently infected cultures after eight cell passages was always temperature-sensitive and of smaller plaque size than the parental virus in chicken embryo cell cultures. Similar results were obtained with virus recovered from L-cell cultures persistently infected with two other velogenic strains of NDV, the Texas-GB and Kansas-Man strains. These results strongly suggest that selection of ts mutants during the persistent infection was not random and played a role in establishment or maintenance of the persistent infection, or both.     

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.     

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.     

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.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures.

Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.     

Pathotyping of Newcastle disease virus with a filamentous bacteriophage

A filamentous phage bearing the peptide sequence TLTTKLY was isolated from a heptapeptide phage display library against a velogenic Newcastle disease virus (NDV). In order to investigate the potential of this specific phage as an immunological reagent in virus pathotyping, an enzyme-linked immunosorbent assay (ELISA)-based method was developed. This method can differentiate the velogenic strains from the mesogenic and lentogenic strains. An equilibrium-binding assay in solution showed that the interactions between the phage and all the NDV strains gave rise to two widely differing dissociation constants (Kdrel). Based upon the first Kdrel values, NDV strains can be classified into two groups; the first comprises the velogenic strains, and the second consists of the mesogenic and lentogenic strains. These results indicate a high degree of correlation between the binding affinities and pathotyping of NDV strains using the TLTTKLY phage.     

用蚀斑法进行麻疹疫苗的病毒滴定

按世界银行中国疫苗项目对麻疹疫苗检定的要求,建立了麻疹疫苗病毒滴定的蚀斑方法。以蚀斑法对Ｈｕ１９１株生产的冻干麻疹活疫苗样品进行了测定。结果发现,Ｈｕ１９１株生产的麻疹疫苗在琼脂和甲基纤维素覆盖下所形成的蚀斑大小及形状不同,但滴度无显著性差异;在室温和３７℃吸附的滴度无显著性差异,但细胞在３７℃生长较好;在本实验条件下,蚀斑滴定和ＣＣＩＤ５０测定的滴度呈正相关,蚀斑滴定相对较敏感;采用国产６孔聚苯乙稀培养板,Ｖｅｒｏ细胞单层,在３７℃吸附,用甲基纤维素覆盖,经结晶紫及甲醛固定染色后计数空斑,操作简便易行,结果特异敏感,重复性好,可用于麻疹疫苗病毒滴定     

Comparative sensitivity of several plaque assay techniques employing TN-368 and IPLB-SF 21AE insect cell lines for plaque variants of Galleria mellonella nuclear polyhedrosis virus

Several plaque assay techniques employing TN-368 or IPLB-SF 21AE cells were evaluated for their usefulness in detecting and distinguishing MP (many polyhedra) and FP (few polyhedra) plaque variants of Galleria mellonella nuclear polyhedrosis virus. Both plaque morphologies were produced using either cell line. Of the overlays tested, the buffered 0.6% methylcellulose overlay yielded the most plaques and was best suited for titration. It was also the easiest overlay to prepare and use. The largest plaques were obtained using either cell line with the 1.0 or 0.75% agarose overlays. Plaque variants were most easily distinguished under 1.0 or 0.75% agarose overlays with IPLB-SF 21 cells. The 0.9% MC overlay was the only overlay which did not allow detection of FP plaques. However, FP plaques were detected using a buffered modification of this overlay. It is concluded that the FP variant of G. mellonella NPV is not a host-dependent phenomenon, and that its detection can be influenced by overlay formulation.     

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.     

Plaquing procedure for infectious hematopoietic necrosis virus

A single overlay plaque assay was designed and evaluated for infectious hematopoietic necrosis virus. Epithelioma papillosum carpio cells were grown in normal atmosphere with tris(hydroxymethyl)aminomethane- or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered media. Plaques were larger and formed more quickly on 1- to 3-day-old cell monolayers than on older monolayers. Cell culture medium with a 10% addition of fetal calf serum (MEM 10) or without serum (MEM 0) were the most efficient virus diluents. Dilution with phosphate-buffered saline, saline, normal broth, or deionized water reduced plaque numbers. Variations in the pH (7.0 to 8.0) of a MEM 0 diluent did not affect plaque numbers. Increasing the volume of viral inoculum above 0.15 ml (15- by 60-mm plate) decreased plaquing efficiency. Significantly more plaques occurred under gum tragacanth and methylcellulose than under agar or agarose overlays. Varying the pH (6.8 to 7.4) of methylcellulose overlays did not significantly change plaque numbers. More plaques formed under the thicker overlays of both methylcellulose and gum tragacanth. Tris(hydroxymethyl)aminomethane and HEPES performed equally well, buffering either medium or overlay. Plaque numbers were reduced when cells were rinsed after virus adsorption or less than 1 h was allowed for adsorption. Variation in adsorption time between 60 and 180 min did not change plaque numbers. The mean plaque formation time was 7 days at 16 degrees C. The viral dose response was linear when the standardized assay was used.     

Plaquing procedure for infectious hematopoietic necrosis virus.

A single overlay plaque assay was designed and evaluated for infectious hematopoietic necrosis virus. Epithelioma papillosum carpio cells were grown in normal atmosphere with tris(hydroxymethyl)aminomethane- or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered media. Plaques were larger and formed more quickly on 1- to 3-day-old cell monolayers than on older monolayers. Cell culture medium with a 10% addition of fetal calf serum (MEM 10) or without serum (MEM 0) were the most efficient virus diluents. Dilution with phosphate-buffered saline, saline, normal broth, or deionized water reduced plaque numbers. Variations in the pH (7.0 to 8.0) of a MEM 0 diluent did not affect plaque numbers. Increasing the volume of viral inoculum above 0.15 ml (15- by 60-mm plate) decreased plaquing efficiency. Significantly more plaques occurred under gum tragacanth and methylcellulose than under agar or agarose overlays. Varying the pH (6.8 to 7.4) of methylcellulose overlays did not significantly change plaque numbers. More plaques formed under the thicker overlays of both methylcellulose and gum tragacanth. Tris(hydroxymethyl)aminomethane and HEPES performed equally well, buffering either medium or overlay. Plaque numbers were reduced when cells were rinsed after virus adsorption or less than 1 h was allowed for adsorption. Variation in adsorption time between 60 and 180 min did not change plaque numbers. The mean plaque formation time was 7 days at 16 degrees C. The viral dose response was linear when the standardized assay was used.     

Rhinovirus Plaque Formation in WI-38 Cells with Methylcellulose Overlay

A sensitive, reliable plaque assay system is described for five rhinoviruses using freshly prepared methylcellulose overlay and human embryonic diploid cells. Circular plaques with irregular edges, 2 mm in size, were formed by rhinoviruses 1A, 2, 6, and 13 after 6 or 7 days of incubation. A fifth rhinovirus, 17, formed a 1- to 2-mm feather plaque after 14 days of incubation. Plaque counts of rhinoviruses 1A and 13 were not affected by varying the pH of the overlay from 6.9 to 7.5. Plaque sizes and plaque-forming unit values of high passage rhinoviruses 1A and 13 were equivalent when tested at 26, 31, or 36 C. The rhinoviruses tested were sensitive to incubation at 40 C or heating at 50 C. Enhancement of plaques was observed when Mg(++) was incorporated into agar overlays, but enhancement did not occur when Mg(++) was added to methylcellulose overlays.     

我国新分离乙型脑炎病毒株毒力特征研究

将分离自不同年代的17株乙脑毒株在小鼠脑内传代,然后将病毒在BHK21细胞单层上观察不同毒株的空斑形成大小形态,小鼠脑内和皮下途径接种观察病毒的毒力,结果显示不同毒株在BHK21细胞上形成的噬斑大小不尽相同,减毒株形成的噬斑最小。所有毒株对小鼠的脑内毒力都很强,病毒滴度高达lg8.0/mL以上,毒株间无明显差异。毒株对9～11g小日龄小鼠的皮下毒力有一定差异但不明显,但对14～16g较大日龄小鼠则差别明显。PFU/LD50的对数值差异除一株(M47株)为8.44外,其余各株差别在3.94～0.45间。本研究结果证明自然界乙脑毒株存在明显的神经外毒力差异,毒力差异与分离年代和病毒基因型无关,但从人体分离到的毒株毒力表现较强。     

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.     

Differentiation of velogenic, mesogenic and lentogenic strains of Newcastle disease virus by multiplex RT-PCR

A multiplex RT‐PCR technique has been developed for differentiation of velogenic, mesogenic and lentogenic pathotypes of Newcastle disease virus (NDV), using a set of three oligonucleotide primers designed from NDV genomic RNA (P1, P2 and P3). The primer pair P1 and P2 generated a RT‐PCR product of 204 bp, only with RNA from velogenic and mesogenic strains, whereas the P1 and P3 generated a 364 bp product only with RNA from mesogenic and lentogenic strains. Thirty four NDV strains, including some reference strains (known pathotypes), NDV field isolates and NDV vaccine strains, as well as other avian virus strains, were tested with multiplex RT‐PCR. All reference strains tested were differentiated in agreement with their intracerebral pathogenicity index (ICPI) values or with the pathotypes known in previous reports. The nucleotide sequence analysis of RT‐PCR products for four NDV strains was fully in agreement with the RT‐PCR characterisations of these strains. The RT‐PCR results of other avian RNA viruses further confirmed the reliability and specificity of this technique. However, the RT‐PCR failed to detect some other avian NDV, which may not originate from chicken. This multiplex RT‐PCR technique is simple and easy to perform. It could be applied not only to determine the origin of NDV, but also may be used diagnostically in molecular epidemiological analysis of ND and for prediction of pathotypes of NDV isolates.     

Plaque Formation by 55 Rhinovirus Serotypes

Plaque production by 55 rhinoviruses in several lines of HeLa cells is reported. Forty-nine types produced macroscopic plaques under the conditions described previously employing 30 mM MgCl(2) and 30 mug/ml of DEAE-dextran in overlay and M HeLa cells. Basically three kinds of plaques were observed: clear large and intermediate sized plaques and small turbid plaques. Five rhinoviruses produced plaques only in a sensitive clonal line isolated from the M HeLa line. Rhinovirus type 4 produced plaques after the pretreatment of cells with actinomycin D. Enhancement of plaque formation by MgCl(2) has been demonstrated to date with 17 rhinoviruses. Some rhinoviruses were enhanced either by DEAE-dextran or by dextran sulfate in the overlay. No inhibitors were found in any of 10 bovine sera tested. Rhinovirus type 2 was visualized inside HeLa cells by electron microscopy and there appeared to be a very high ratio of physical particles per infectious unit of virus.     

Sequential passages of human rotavirus in MA-104 cells

Starting with a small amount of diarrheal feces containing human rotavirus (HRV), we succeeded in propagation of the virus using the roller culture technique with MA-104 cells. Furthermore, we made a successful adaptation of HRV to a stationary culture and developed a plaque assay for the cell culture-adapted viruses. The 3 culture-adapted virus isolates, KU, YO, and 44 produced plaques (about 0.5-1.0 mm in diameter) under the overlay medium consisting of 0.6% purified agar, 3 micrograms of acetyl trypsin/ml and 50 micrograms of DEAE-dextran/ml. Subsequent plaque purification resulted in the formation of clear, larger plaques. It was shown from the results of cross neutralization tests using the fluorescent focus reduction method that the three culture-adapted HRV isolates were clearly different antigenically from ovine rotavirus (NCDV) and, further, that a noticeable difference in antigenicity also existed among the HRV isolates.     

Host influence on the characteristics of Venezuelan equine encephalomyelitis virus

Heydrick, Fred P. (Fort Detrick, Frederick, Md.), Ralph F. Wachter, and Henry J. Hearn, Jr. Host influence on the characteristics of Venezuelan equine encephalomyelitis virus. J. Bacteriol. 91:2343-2348. 1966.-Alterations in plaque size, virulence, and lipid content of Venezuelan equine encephalomyelitis (VEE) virus were examined for possible interrelationships among these properties during 10 serial passages in embryonated eggs, suckling mice, chick embryo fibroblasts, and L cells. The chick embryo host maintained the same large-plaque and virulence properties of the virus through 10 passages as seen in the original seed. Passage of virus in either L cells or chick fibroblasts rapidly produced populations that were, in the main, intermediate with respect to plaque size and virulence. Passage of virus in suckling mouse brain yielded populations that were intermediate with respect to plaque size only. The nature of the lipid of the virus, in terms of the ratio of petroleum ether-soluble to -insoluble lipid, changed after only one passage in all systems except in chick embryos. Nine additional serial passages failed to enhance these changes in viral lipid, suggesting that the decrease in the large-plaque and virulence properties was not directly associated with changes in lipid content.     

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.