Richard I. Carp

A comparison of the replication cycles of simian virus 40 in human diploid and African green monkey kidney cells

Abstract Simian virus 40 (SV40), which is capable of inducing transformation of human diploid cells, replicated to only a limited extent in WI-38 cultures and failed to cause any cytopathic changes. Less than 3 infectious particles were produced per cell in WI-38 cultures. In contrast, SV40 replicated extensively in primary African green monkey kidney cells (GMK) and in an established cell line (BS-C-1) obtained from GMK, with 100 and 50 infectious particles produced per cell, respectively. In addition, the virus caused extensive cytopathic changes in these cells. A comparison of the infectious cycle in a lytic (BS-C-1 and GMK) and in a transformable (WI-38) system, revealed that there was no difference in virus adsorption or entrance into eclipse in the two systems. Furthermore, there was no suppression of ribonucleic acid (RNA) or protein synthesis in WI-38 cells, nor in the stationary phase or replicating GMK cells following infection with SV40. The only early function that differed in the two systems was the production of SV40 Induced Complement Fixing Antigen (SV40 ICFA). Less than 3% of the WI-38 cells in a culture infected with a high multiplicity of virus developed SV40 ICFA during the first 7 days postinfection, whereas 80–100% of infected GMK cells were positive for ICFA 3 days after infection. There were marked differences in the infectious cycle of SV40 in replicating and stationary phase GMK cells. The yield of virus per cell, the development of cytopathic effects, and the production of SV40 ICFA were comparatively low in replicating cells; cells infected while replicating continued to multiply despite infection and have been subcultured 14 times since the addition of virus.

Altered scrapie infectivity estimates by titration and incubation period in the presence of detergents.

During experiments on the purification of scrapie infectivity, changes were found in the dynamics of scrapie titration. After exposure to detergent, infectivity estimates by both endpoint titration and incubation period were altered. The addition of detergent to the diluent used in titration resulted in at least a 100-fold increase in the infectivity estimate. This suggests that the amount of scrapie in a sample, as measured by serial dilution and titration, may be underestimated to different extents, depending on the biochemical milieu of the inoculum. Membrane fractions treated with detergents before dilution exhibited longer incubation periods than untreated fractions for the same number of infectious units of scrapie. This demonstrates that detergent treatments and possibly other biochemical manipulations can cause changes in the response of the host to the inoculum that are not detectable if incubation periods alone are used to estimate scrapie titre.

The effect of actinomycin D on the transcription and replication of simian virus 40 deoxyribonucleic acid

Abstract In green monkey kidney (GMK) cells infected with simian virus 40 (SV40), the synthesis of virus, viral deoxyribonucleic acid (DNA), “late” virus-specific ribonucleic acid (RNA), and viral coat protein are suppressed by actinomycin D (0.5 μg per milliliter of medium). In contrast, the drug does not inhibit the synthesis of tumor (T) antigen or of early virus-specific RNA. Thymidine kinase induction in SV40-infected cells is decreased but not eliminated by actinomycin D. Early virus-specific RNA accumulates in the presence of actinomycin D to levels comparable to the total virus-specific RNA synthesized in the absence of the drug.

Investigations of the reproductive capacity temperature marker of polioviruses

Abstract 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°.

THE POLIOVIRUSES OF MAN

Of no other animal virus-with the exception of influenza-do we know as much as of the three types of Poliovirus horninis. Fifty years of experimental research dating from the time of Landsteiner, as well as numerous international conferences and monographs, have contributed to our present knowledge. Indeed, articles on poliomyelitis are published in such profusion that a journal of poliomyelitis abstracts is printed each month. The task assigned to us of providing a brief summary of knowledge of the polioviruses thus requires a style more epigrammatic than deep and more eclectic than broad. Although “no epigram conveys the whole truth,” we are justified in such an approach, as in many respects the polioviruses can serve as standards for comparison with other viruses to be discussed later in this conference. In this review an unhistoric point of view will be taken, with emphasis on current data rather than on the development of ideas.

Mutation of type 3 poliovirus with nitrous acid.

Abstract The incubation of RNA from type 3 poliovirus (W-Fox strain) with nitrous acid caused a decrease in the average plaque size and an increase in the proportion of “small-plaque” formers in the surviving population. These “small-plaque” mutants bred true after passage in tissue culture. A reduced burst size of the treated particles was found to be the probable cause of the small plaque size. Virus isolated after the passage of several of the mutant plaque progeny pools in the human intestine showed a marked alteration from the virus fed in plaque size and reproductive capacity temperature (rct) markers (from the small, rct 40− character of the fed virus to the larger, rct 40+ character of the virus isolated). The incubation of W-Fox RNA with nitrous acid did not change the rct 40− character of the virus.

A new test of the reproductive capacity temperature marker of poliovirus: the limited thermal exposure test☆

Abstract The exposure of infected monolayers to increased temperatures during the early part of the incubation period was found markedly to inhibit plaque formation by viruses that replicate poorly at 40°, without reducing the number of plaques produced by strains able to grow at this temperature. This observation was used as the basis for the development of a sensitive test for the “rct” marker. The test, which has been termed the limited thermal exposure test, was applied to numerous test strains. The results were found to agree well with those obtained using other tests of the “rct character.” Inhibition of plaque formation by attenuated strains also occurred at 39°, a result indicating that the LTE test is less sensitive to variations in incubation temperature than other tests. Following the exposure of infected monolayers to increased temperatures for short periods, virulent, rct 40+ strains frequently showed an increase in plaque counts.

THE EFFECT OF DEUTERIUM OXIDE ON VIRUS‐HOST INTERACTION*

The effect of D/sub 2/O on the growth of E. coli and a number of mammalian stable cell lines has been investigated. As the D/sub 2/O concentration was increased, the cells of all the species tested appeared larger, showed an increase in water content and dry weight, and had a slower growth rate. Cytological investigations showed an increase in the number of multinucleated cells and a moderate increase in sudanophilic material. In general, cells grown in D/sub 2/O exhibited cytological characteristics similar to those observed in giant cells produced by irradiation. The effect of D/sub 2/O on bacteriophage replication was examined. The burst size of T5 was somewhat reduced in deuterated E. coli, but the burst size of T7 was significantly increased. These differences might be explained by the fact that whereas the bulk of T5 DNA is derived from the medium, most of the T7 DNA is derived from the host. With increased size of the host, more DNA and protein production might be expected. Whether or not deuteration causes an inhibition of the uptake of various substrates from the medium is under investigation. Studies of the multiplication of poliovirus in deuterated HeLa and monkey kidney cells also showedmorexa0» a marked incresse in burst size. A possible variant of CHAT (attenuated type 1 poliovirus) was isolated from virus progeny grown in deuterated monkey kidney cells. The reason for the increased burst size in deuterated cells is not clear at the present time. Similar increases in burst size were observed in studies of viral replication in x ray treated giant cells. It is possible that the enlargement of cells grown in D/sub 2/O is the underlying factor in the observed increase of poliovirus burst size. (auth)«xa0less

Modification of an Attenuated Type 2 Polio Vaccine Following Passage at 23°C.

Summary The modification of a type 2 attenuated poliovirus strain is described. Starting with the mouse adapted TN strain, passage and clone selection at low temperatures of incubation were employed to yield virus (the W-II strain) with reduced pathogenicity for monkeys and good stability during human intestinal passage.

Effects of deuterium oxide upon poliovirus multiplication

The effects of deuterium oxide on the multiplication of CHAT, an attenuated type of poliomyeliths virus, was studied in cells of HeLa and of monkey kidney cells in primary cultures. Yields of virus obtained from deuterated cells were consistently higher than those obtained from controls. The incorporation of deuterium oxide in the growth media resulted in an increase in the average plague size of polio virus. (C.H.)