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We have investigated the replication and persistence of human papillomavirus (HPV) type 6 and 11 DNA in cultured cells derived from laryngeal papillomas, with paradoxical findings. Measured by bromodeoxyuridine incorporation into heavy/light DNA separated on a cesium chloride gradient, viral DNA replicates in both primary and secondary cells. The ratio of the fraction of replicated viral to replicated cellular DNA was equal to or greater than 1 in all but one case and was closer to 2 in primary cells. Despite this efficient replication, HPV DNA is rapidly lost from the cells with passage. We propose that infected cells, or those with a high HPV copy number, show a selective decrease in plating efficiency compared to uninfected cells or those with a low copy number, which explains the loss of HPV DNA with repeated passage.

A DNA-containing bacteriophage ∅Cp34 was isolated for Caulobacter . Phage ∅Cp34 consists of a prolate cylindrical head with a flexible and noncontractile tail. The host range is limited to Caulobacter crescentus and Caulobacter vibrioides . A remarkable property of phage ∅Cp34 is the requirement of active flagella for infection (flagellotropy). The flagellotropy of phage ∅Cp34 was shown by the following observations: (a) reduced plating efficiency on the flagella-less mutant CBM-2 and recovery to the normal one upon CBM-2 reversion to the motile cell; (b) low ∅Cp34-adsorbing capacity in the mutant CBM-2 and subsequent recovery of such capacity when the mutant CBM-2 reverts to the motile cell; (c) low ∅Cp34-adsorbing capacity in the wild-type cells sheared to remove flagella and subsequent recovery of such capacity when the flagella are regenerated in the sheared cells; (d) reduced plating efficiency of phage ∅Cp34 and absence of ∅Cp34 adsorption and eclipse when flagellar motion is blocked by high viscosity; (e) no attachment of phage ∅Cp34 onto isolated flagella. Phage ∅Cp34 was observed under an electron microscope to be attached onto flagella and adsorbed at polar sites proximal to the single flagellum in the swarmer cell. 32 P-labeled phage ∅Cp34 was used to specifically assay newly formed flagella in ∅Cp34-resistant mutant and extended for monitoring the flagella formation in the synchronous cell cycle of Caulobacter .

Electron microscopic length measurement of Eco RI limit digest products of mature P2 DNA reveals four fragments, A, B, C and D, of defined length. Under our standard conditions, the efficiency of plating of P2 on an RI-carrying strain is 7–10%. An exception is the deletion mutant P2 del2 having a plating efficiency of 30–40%. Electron microscopy of Eco RI digests of P2 del2 DNA shows that del2 removes the middle RI-sensitive site. Since the location of del2 is precisely known from previous work, and the two end segments are sometimes observed joined through their cohesive ends, it is possible to locate the three sites (leftmost, middle, and rightmost) at 10.5, 47.5, and 79.9% from the left end of P2 DNA, respectively. The rightmost site is most probably within cistron B. A mutant, P2 del2 sr1 , was isolated which has a plating efficiency of 70–90% on the RI-carrying host. Electron microscopy of P2 del2 sr1 DNA and phage crosses show that mutation sr1 makes the rightmost site resistant to Eco R1. The sr1 mutation, when separated from del2 , gives a plating efficiency of 20–30% on the RI-carrying host. It would seem that the three sites do not contribute equally to the lowering of the efficiency of plating.

Mild treatment of 3T3 cells with low concentrations of Clostridium perfringens neuraminidase and phospholipase C resulted in inhibition of interferon production. The enzymatic digestion did not affect the viability and plating efficiency of these cells. Treatment of the cells with concentrations of neuraminidase and phospholipase C, which caused complete suppression of the rI n · rC n response did not affect binding of the inducer to these cells. Thus, the antiviral response and cellular binding properties of rI n ·rC n can be dissociated. The rI n ·rC n inducible interferon production of neuraminidase- and phospholipase C-treated cells was restored upon prolonged incubation in media. The observed inhibition was not specific for 3T3 cell systems; human fibroblast cells responded similarly. Finally, radioactive DEAE-dextran was synthesized and its binding to 3T3 cells was studied under conditions which potentiate interferon induction. While the binding of rI n ·rC n is greatly enhanced in the presence of DEAE-dextran, the binding of DEAE-dextran was independent of the presence of rI n ·rC n .

Infectious ribonucleic acid (RNA) from foot-and-mouth disease virus, type A, was examined as to its method of preparation, stability, and plating efficiency on calf-kidney cultures. The best yields of RNA were obtained with cold phenol at low ionic strength in the presence of EDTA and sodium dodecylsulfate. One extraction with phenol was sufficient to destroy all ribonuclease (RNAase)-resistant virus particles and gave the maximum yields of RNA. The isolated RNA was completely stable to storage at −196°C but not at 4°, −12° and −50°C. It was more stable than the virus itself to acid and alkaline conditions from pH 3.0 to 11.5. When acidified to pH 4.1 in the absence of RNAase, the virus did not appear to split off infectious RNA. There was a linear relationship between plaque counts in tissue culture and the dilution of RNA applied, and replicate platings produced a Poissonian distribution of plaque counts. The plating of RNA was considerably more efficient when small-sized (i.e., 0.1 ml) inocula were used than when the same amount of RNA was applied in larger volumes. Under such conditions the infection of culture cells appeared to be rapid, i.e., as many plaques were formed when the agar overlay was applied 1 minute after plating RNA as when applied at later times.

Poliovirus grown in 4 m M 5-fluorouracil (5-FU) at an optimum temperature (36°) becomes more “heat defective”; that is, the ratio of plaques formed at supraoptimal temperatures to plaques formed at 36° (“plating efficiency”) is decreased by a factor of 3–10. The plating efficiency at an infraoptimal temperature (30°) is unchanged. These findings are true for strains with very different plating efficiencies at both high and low temperatures. The enhanced heat defectiveness is retained after subculture in absence of 5-FU: since selection of preexisting heat-defective ( hd ) mutants is shown to be unlikely, it is concluded that 5-FU is highly mutagenic for poliovirus when judged by the character of plating efficiency at high temperatures. This is confirmed by the plaque isolation of hd mutants, which comprise at least 10% of the progeny of a nondefective ( hd + ) strain grown in 1 m M 5-FU; normal hd + progeny contain 1–2% of hd mutants. Poliovirus growth is inhibited by 5-FU to an extent which depends on the intrinsic heat defectiveness of the strain: the lower the plating efficiency at high temperatures, the poorer the growth in 5-FU at 36°.

The inhibitory effect of neutral red on plaque formation by foot-and-mouth disease virus, type A, on primary bovine calf-kidney tissue cultures has been studied. The adsorption of virus is complete within 20 minutes at 37°. However, 70 additional minutes of incubation are required to observe maximal titers when 1:10,000 dilution of neutral red is used in the agar nutrient medium. Decreasing the neutral red concentration decreases the incubation time necessary for maximal titers. By appropriately diminishing the concentration of this stain in an agar overlay, the same plating efficiency is obtained as when staining is withheld until after the necrotic areas have developed. It is concluded that some early stages of virus replication are subject to inhibition by neutral red through the action on the host cell, and that after this stage is past, the stain has no detectable effect on further acts of replication.

A quantitative in vitro assay has been developed for avian sarcoma viruses capable of converting rat embryo fibroblasts. The physical properties of the agent capable of converting rat cells are the same as those of the virion of avian sarcoma virus. Avian sarcoma virus recovered after passage through converted rat cells is genetically altered having increased plating efficiency on rat cells and altered antigenicity. The plating efficiency on rat cells is not correlated with the type-specific antigen of the virus. Phenotypically mixed virus has plating efficiencies on duck and rat cells like virus with the same genome, not like virus with the same envelope. Treatment of rat cells with inactivated Sendai virus does not greatly increase the plating efficiency of avian sarcoma viruses on these cells. Therefore, passage of avian sarcoma virus through rat cells results in virus that is genetically altered in two separate phenotypic properties. A model to explain these results involving recombination between the avian sarcoma virus and the host genome is proposed.

Differences in properties between normal hamster embryo cells and clonal lines of hamster embryo cells transformed with polyoma virus provided means of selecting for the growth of colonies of transformed cells in the presence of many normal cells. In reconstruction experiments where selective techniques were applied to artificial mixtures of small numbers of transformed cells and large numbers of normal cells, transformed cells could be detected at frequencies of 1 cell in 104 normal cells to 1 cell in 105 normal cells, depending upon the particular transformed line of cells used. Cells of one line, termed line Li, of particular interest because its properties were between those of normal cells and cells of the other transformed lines, could not be satisfactorily detected by any of the selective techniques employed. By means of reconstruction experiments, techniques were developed which succeeded in detecting cells of this line at a frequency of one cell in 104 normal cells. When the selective techniques were applied to cultures immediately after adsorption of virus, transformed cells were detected and selected for by some of the techniques, particularly those that were most successful for line Li. Thus new properties, such as a high relative plating efficiency in media containing low concentrations of serum, on which the techniques depend, are conferred upon cells very soon after infection with virus. The selective techniques succeeded in measuring frequencies of transformation too low to be measured by a nonselective colony assay, and thus provide the basis for an assay for transformation of considerably increased sensitivity.

The proportion of hamster cells transformed in vitro was determined after exposure to various doses of polyoma virus. Preliminary experiments were made to characterize the transformed cells; the plating efficiency was determined with different types and numbers of irradiated feeder cells, and in the presence of excess normal hamster cells. Virus was adsorbed to hamster cells in suspension, and known numbers of cells were plated. Subsequently, colonies of transformed and normal cells were counted. After exposure to 96 PFU per cell the proportion of transformed colonies was 10−4 of original cells plated. This indicated transformation of between 10−2 and 10−3.7 of the original cells, depending on the plating efficiency immediately after virus adsorption. In various experiments involving exposure to 0.13 to 96 PFU per cell, the number of transformed colonies was directly related to virus dose. The size of some transformed colonies after 7 days suggested that there was little delay between virus infection and alteration of the hereditary characteristics of the cell.

Type 2 adenovirus growing in KB or other human cells has an absolute requirement for arginine in the medium. Without arginine, there is no net synthesis of virus. Of the amino acids tested, arginine is the only one for which there is such a requirement. Established lines of human cells are often contaminated with pleuropneumonia-like organisms (PPLO), and these organisms have the capacity of rapidly depleting the available arginine, thereby lowering the efficiency of plating of various adenoviruses. These fluctuations in plating efficiency can be eliminated by curing the cells of PPLO infection.

A plaque assay for foot-and-mouth disease virus, type A, was developed in primary bovine kidney cultures, and the kinetic course of virus reproduction was established. The effect of environmental variables on plating efficiency was studied. Exposure of virus to cell layers for 90 minutes at 37° or 15 minutes at 43° prior to overlaying with agar and incubating for an additional 72 hours at 37° gave maximal plaque counts. Temperatures of 26° or 29° maintained during the adsorption period did not produce maximal numbers of plaques. Plating efficiency depended upon the volume of diluent added to virus samples before exposure to cell layers. For example, identical amounts of virus applied to cultures in volumes of 0.1, 0.4, and 6.4 ml produced plaques in the approximate relationship of 60:30:10. Virus diluent should contain serum. Without serum, calcium and magnesium ions were not fully effective. Washing cultures with phosphate-buffered saline before and after exposure to virus was not required, and it was shown that plaques developed only from infections initiated before agar was applied. Frequency distribution and dilution experiments showed that plaques were initiated by single virus particles. Where mean counts of replicate platings ranged from 47 to 66, twice the standard deviation of the means ranged from 16 to 31%. In virus reproduction experiments progeny appeared at 2.5 hours. At 11.5 hours there were 108.6 plaque-forming units (PFU) per millitier corresponding to a release from each cell of approximately 370 PFU.

Changes of the sequence or of the relative duration of combined high and low pH incubation affect the frequency with which transformed colonies arise in cultures of polyoma virus-infected BHK21 cells. In general, high pH incubation increases the number of transformed colonies but reduces the plating efficiency of nontransformed infected cells while low pH incubation has opposite effects. However the effect of pH on transformation is independent of its effect on the plating efficiency of nontransformed cells. Low pH incubation also increases the ratio of “mixed” to “pure” transformed colonies. Two-day “pulses” of low pH reduce the number of transformed colonies, but this “pulse” effect diminishes progressively with time, apparently because of a progressively diminishing inhibition of formation of “pure” transformed colonies. The effect of pH on plating efficiency is greatest on the first day post infection and then rapidly declines at a different rate than the effect on transformation. The data confirm that “mixed” transformed colonies arise continuously after infection and suggest that “pure” transformed colonies also continue to arise for 3–4 days after infection. They further indicate that the development of “mixed” and “pure” transformed colonies is affected differently by high and low pH. The concept of “nascent” transformed cells as opposed to established transformed cells is discussed.

Lysogenic cultures with derepressed λN − prophages contain 20–25 λ genomes per bacterial genome. Such culture can be maintained indefinitely and the bacteria produce colonies with a distinctive phenotype. Wild-type imm λ and both imm 434 and imm λ vir generally fail to form plaques on these polylysogens. However, imm 21 and imm λ c 17 mutants form plaques with an efficiency of 40–50%. Three different factors contribute to low efficiency of plating of imm λ and related phages in λN − polylysogens. Superinfecting imm λ is subject to inhibition of expression of its N gene, mediated by Tof product produced by the prophages. In the case of imm 434, or the vir mutant of imm λ, plating efficiency is very low because the genome of a superinfecting phage has a low probability of appearing in the burst of progeny phage. Competition of prophage genomes for a transcription factor is postulated to account for the failure of about 50% of heteroimmune superinfecting genomes to express gene N . All these effects can be overcome by increasing the multiplicity of infection.

The transformation of primary rat cells into established cell lines by simian virus 40 has been monitored using the different restrictive assays of colony formation in sparse culture, dense colony or focus formation on a confluent cell sheet, and colony formation in semisolid medium. Primary embryonic rat cell cultures are considerably less susceptivle to infection and subsequent transformation than the established mouse 3T3 cell line or later in vitro passages of rat cells. These embryonic cells show a stage-specific susceptibility to transformation but not to infection with a maximum susceptibility achieved at the 15th to 16th days of gestation. All transformed cell lines derived by SV40 infection of primary rat cells express viral T antigen as detected by immunofluorescence, though they differ greatly in their plating efficiency in semisolid medium containing methycellulose. Only the assay of colony formation in semisolid medium selects directly for transformants which plate well in that medium while all assays appear to select for cell lines containing viral T antigen.

A chikungunya (CHIK) virus-resistant line of Singh's Aedes albopictus (SAAK) cells released spontaneously two kinds of plaque-forming agents which could be distinguished by the plaque size on a sensitive clone C6/36 of another A. albopictus cell line (SAAR). Release of the large plaque (Lp) and the small plaque (sp) viruses into the culture medium occurred at different rates when the SAAK cells were subcultured. The Lp virus was removed from the SAAK cells by subculturing or cloning in the presence of anti-CHIK serum. Such Lp(-) cells continued to produce the sp virus, but were permissive for CHIK virus. The plaque-purified Lp virus, but not sp virus, interfered with the growth of CHIK virus in the C6/36 cells. The Lp virus resembled in physicochemical and serological properties CHIK virus grown in C6/36 cells. However the Lp virus was not pathogenic for suckling mice, was heat labile, and showed higher plating efficiency on the C6/36 cells than on BHK21 cells. The data indicate that the Lp virus is a mutant of CHIK virus and is responsible for the CHIK virus resistance of the SAAK cells.

Thymidine kinase-deficient syrian hamster cells were cotransfected with recombinant plasmids containing the thymidine kinase (TK) gene of Herpes Simplex Virus Type 1, and either intact or partially deleted SV40 T antigen-coding genes. The transformants were selected by their ability to grow in gHAT medium. After selection and cloning, the TK-positive transformants that also expressed T antigen were tested for the extent of their transformation with respect to a number of characteristics, which included saturation density, ability to grow in soft agar, resistance to butyrate and to dibutyryl-cAMP, and plating efficiency. The combined results of these various tests indicate that cells containing partially deleted SV40 T antigen-coding genes are less transformed than cells containing an intact SV40 T antigen-coding gene. However, the amounts of T antigen are lower in cells transformed by deletion mutants than in cells transformed by wild-type T antigen-coding gene. Our data indicate that both the quantity and the quality of T antigen may be important in determining the degree of transformation in Syrian hamster cells.

The state of the SV40 DNA in a stable carrier system of At 72 human glioblastoma cells was examined by Southern blot hybridization analysis. At a sensitivity of 0.1 viral genomeequivalents per cell, we detected only free, apparently nondefective, viral genomes. However, when we overexposed our autoradiograms or examined cloned cell populations, integrated viral sequences were observed. Futhermore, aberrant forms of free viral DNA were seen as well. Four clones, isolated at 15 weeks, produced T antigen and displayed enhanced saturation density and plating efficiency characteristic of SV40 transformation. None of these clones produced capsid proteins or infectious virus, even upon fusion with CV-1 cells, Viral DNA in the clones ranged from 0.5 to 50 equivalents per cell, on the average. Two of the Week-15 clones contained a similar (but not identical) predominant truncated SV40 sequence which was present both in a free state and integrated at a single major site in a reiterated head-to-tail array. These clones also contained other minor integrated sequences. Another Week-15 clone contained viral sequences integrated at two major sites as well as heterogeneous free DNA. Only free aberrant DNA was detected in the fourth Week-15 clone. Seven of eight clones isolated at 23 weeks produced no infectious virus or T antigen. No viral DNA was detected in those clones. The eighth clone did produce infectious virus and contained a predominance of free viral DNA. All of the clones were susceptible to superinfection with wild-type SV40, although less so than uninfected At 72 cultures.

We explored host cell and viral factors which influence the susceptibility of lymphocytes to immortalization by the Epstein-Barr virus (EBV). We found that immortalization by the B95-8 strain of EBV follows “one-hit” kinetics. With a limiting dilution technique, approximately 1 in 20 virus particles is transforming when assayed on human umbilical cord leukocytes (HUCL). Mixed mononuclear leukocytes from adult human and marmoset blood are 100- and 1000-fold, respectively, less sensitive to immortalization than HUCL. Pretreatment of cord blood leukocytes with phytohemagglutinin results in a 50% increase in their transformability. Lipopolysaccharide (LPS) from Escherichia coli increases the number of transformation events observed by 300–500%. LPS appears to enhance transformation by mechanisms other than stimulation of cellular DNA synthesis. A transformed center assay employing either autochthonous lymphocytes or human placental cells as feeders was used to determine the number of umbilical cord cells which were immortalized. At multiplicities of infection of 30–100 particles per cell, multiplicities which saturate nearly all the susceptible cells, approximately 1 in 200 to 1 in 500 mixed mononuclear cells transformed. When the mixed population of cells was depleted of T lymphocytes, there was a fourfold increase in the number of transformed cells. When correction is made for the plating efficiency of transformed cord blood leukocytes, it is calculated that at least 10% of virus-exposed cells establish permanent lines. This number is probably still an underestimate since we have not yet used a population which is composed entirely of susceptible cells.

Cells from three different mouse strains were transformed in vitro with 2 mutants of SV40 as well as with wild-type virus. SV40 virus could be recovered from transformed ALN and AKR cells by cocultivation with susceptible cells, but virus production occurred much sooner when cells were allowed to form heterokaryons with UV-inactivated Sendai virus. 3T3 transformed cells which did not yield virus by simple cocultivation did yield virus by the cell-fusion technique. SV40 virus recovered from transformed cells was examined and compared with the original transforming virus for three genetic markers: (a) plaque size in primary AGMK, (b) growth at 40°, and (c) plating efficiency and plaque size in an established AGMK cell line, CV-1. In all cases, the virus recovered possessed the markers of the original parental virus.

Some of the factors involved in the interaction between SV40-induced, virogenic hamster tumor cells and indicator cells were investigated. The number of detectable virogenic tumor cells was found to depend on their plating efficiency. Demonstration of virogenicity required viability of the tumor cells as well as direct contact with the indicator cells. This interaction could be enhanced by the presence of ultraviolet-irradiated Sendai virus. The tumor cells retained their virogenic character following prolonged passage in the presence of 10μg/ml of cytosine arabinoside.

The effects of increased incubation temperature upon the replication cycle of an rct 40 − and an rct 40 + strain were studied. It was found with CHAT, the rct 40 − strain, that the higher temperatures did not inhibit the rate at which the virus was absorbed or at which it entered into the eclipse phase of its reproductive cycle. The production of infectious CHAT-RNA by infected cells, however, was found to be markedly reduced at 40°C as compared to 37°. With Mahoney, an rct 40 + strain, similar amounts of infectious RNA were produced at the two temperatures. The plating efficiency of Mahoney was improved if infected monolayers were incubated for a short time at the higher temperature; a result which may be correlated with the observation that a temperature of 40° appears to stimulate the entrance of virus into the eclipse phase. A sensitive test of the “rct” marker was developed, employing an increased incubation temperature for a limited period of time during the early part of plaque formation. The effect of deuterium oxide upon the replication cycle of poliovirus was studied. Of particular interest was the fact that CHAT virus replicated and formed plaques at 40° if infected cells were deuterated. The progeny of CHAT plaques which developed at 40° showed an increased tendency (compared to parental virus) to form plaques after limited exposure of infected monolayers to 40°.

A culture of HeLa cells persistently infected with hemadsorption type 2 virus (HeLa/HA2) was shown to have a similar plating efficiency to that of uninfected HeLa cells, demonstrating the infection was not pathologic. Each of the cells from derived clones contained virus antigen when examined by immunofluorescent staining. When mixtures of HeLa/HA2 and normal HeLa cells were seeded in a single dish in the absence of antiviral serum, and allowed to form clones, the immunofluorescent positive and negative clones were easily differentiated. The ratio of the antigen bearing to nonbearing clones was very close to that of the number of HeLa/HA2 and normal HeLa cells in the original mixture, indicating that the virus antigen of HeLa/HA2 cells could not transfer to the normal HeLa cells. When the HeLa/HA2 clones were grown in medium containing specific antiviral serum, the serum did not affect the formation of the virus antigen in the cell. Therefore, the persistent infection was maintained by transfer to daughter cells during cell division.

Lysis inhibition has been demonstrated with a small-plaque mutant of T5, designated M12. This appears to be the first example of the phenomenon outside the T-even system. M12 and T5 appear identical in host range, adsorption rate, serum neutralization, and injection rate, but differ in minimal latent period in single infection (45–50 minutes for M12 as against 37 minutes for T5) and in plating efficiency under various conditions. Evidence of lysis inhibition in multiply infected cultures was obtained with M12, but not T5, by observations on the clearing of turbid cultures, one-step growth experiments, and microscopic examination. The best demonstrations in one-step growth experiments were obtained when young bacterial cultures (Escherichia coli strain F, smooth derivative of FCb) were multiply infected with M12 in a single input (multiplicity of 6–8 adequate) and further incubated undiluted for 30–40 minutes, or diluted earlier into sonicated extracts of B 5 . The latter do not affect the lysis of cultures singly or multiply infected with T5 or singly infected with M12. One-step growth experiments suggest multistep release of phage in lysisinhibited cultures. A possible explanation, actual multiplication of the infected bacteria, received no support from microscopic studies.