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Viruses are commonly carried by monkeys and may appear as contaminants in cell cultures of their tissues, especially the kidney. Included among these are B virus, foamy agent, measles-like virus, hemadsorption viruses, LCM virus, arborviruses, and a wide variety of miscellaneous viral agents (1-7). Hull et al.(2,3) have studied extensively viruses encountered in "normal" monkey renal cell cultures. Hull called them simian or SV viruses and classified them into 4 groups. based on the kind of cytopathic change induced in monkey kidney cell cultures infected with the agents. Twenty-eight of these viruses were precisely separated serologically into types and, additionally, 24 unidentified viruses were recorded. Malherbe and Harwin (4) distinguished 7 distinct types among the simian agents or SA viruses they recovered from vervet kidney materials.

During the past 2 years, our virus research group has repeatedly encountered a new simian virus of rhesus and cynomolgus monkey kidney origin. This agent was unique among simian viruses studied hitherto since it grew but did not cause a cytopathic effect in the rhesus or cynomolgus kidney cell cultures from which it was derived. Instead, it grew and caused marked cytopathic changes in cell cultures of a heterologous species, i.e., the green monkey, Cercopithecus aethiops obtained from Equatorial East Africa [grivet, according to Sanderson (8)]. This agent has been called the "vacuolating virus" by us because of the prominent cytoplasmic vacuolation seen in infected cell cultures. Dr. Hullt suggested that this agent be given the "official" designation of S.V.40 in C.P.E. group 4. The discovery of this new virus, the vacuolating agent, represents the detection for the

• The first such agent was recognized by Dr. L. McClelland in the course of safety testing of vaceines.

↑ Personal communication.

first time of a hitherto "non-detectable" simian virus of monkey renal cultures and raises the important question of the existence of other such viruses. The present report summarizes the current information available concerning the vacuolating agent. Certain of these data have been presented at the Second International Conference on Live Poliovirus Vaccines (9).

Materials and methods. Tissue cultures. Primary cell cultures were prepared from trypsinized cell suspensions and were grown in Melnick's lactalbumin-yeast extract medium (10) containing 2% heat-inactivated calf serum. All line cell cultures were grown in Eagle's basal medium (10) containing 10% heat-inactivated horse serum. During virus propagation, all cell cultures were maintained using medium 199(10) containing 2% heatinactivated chicken serum and at initial pH 7.6. All culture fluids contained 100 μg of penicillin and 100 units of streptomycin per ml.

Propagation of vacuolating agent. The virus grew readily in cell cultures of grivet monkey kidney with production of cytopathic change. Such cultures were used routinely for the work described in this report. For ordinary purpose, the virus was passed in an initial dilution of 10-1 of infected tissue culture fluid using 0.2 ml of inoculum per 2.0 ml of maintenance medium. After 4 to 7 days incubation, at a time when at least 50% of the cell sheet showed cytopathic change typical of that of the vacuolating agent, the cultures were frozen and thawed once and the fluid was harvested. The virus was stored frozen in the dry ice refrigerator.

Virus titrations and serum neutralization tests. All virus titrations were performed using serial 10-fold dilutions of virus. The inoculum dose was 0.2 ml per roller tube culture containing 2.0 ml of maintenance medium. The end point was the highest initial

of 25% or more of the cells in the cultures after 7 to 10 days incubation. In the neutralization tests, serial 2-fold dilutions of serum inactivated at 56°C for 30 minutes were incubated for one hour at 37°C with an equal volume of virus diluted to contain about 200 TCD per 0.2 ml. The inoculum was 0.2 ml of the virus-serum mixture and the end point was the highest initial dilution of serum which effected complete suppression of cytopathic change in cultures observed 7 to 10 days following inoculation.

Preparation of antiserum against the vacu olating agent. Young adult rabbits were injected intravenously with 1.0 or 2.0 ml of infected grivet monkey kidney culture fluid plus 1.0 ml of infected fluid emulsified in Arlacel-Drakeol adjuvant administered intramuscularly in a single dose or intracutaneously in divided doses. A final 1.0 ml dose of infected fluid was administered intravenously 3 weeks later and the animals were bled one week thereafter.

Results. Recovery of strains of vacuolating virus. Vacuolating virus was recovered from "normal" cell cultures of rhesus and cynomolgus monkey kidney and from the seed stocks of a variety of viruses which had become contaminated with the vacuolating virus during passage in cultures of rhesus and cynomolgus monkey kidney.

The vacuolating virus was recovered from contaminated virus seeds by passage of the virus seed, in the presence of its homologous antiserum, in cell cultures of Cercopithecus aethiops or grivet monkey. Under these conditions, the vacuolating virus "broke through" and was identified in serum neutralization tests using vacuolating virus antiserum prepared in rabbits. Recovery of the virus from "normal" rhesus or cynomolgus cell cultures was accomplished by simple pas sage, in grivet cell cultures, of the fluid from the rhesus or cynomolgus cultures which had been held for 7 to 10 days with maintenance medium. Vacuolating virus isolates were identified in serum neutralization tests using rabbit antisera prepared against prototype strains.

Strain

776 175 8 207

Vires

8211 Sabin, type 2, live attenuated oral potio vaccine, strain

P712 8215 Sabin, type 3, live attenuated oral polio vaccine, strain Leoa

953 Uninfected "normal" rhesus monkey kidney cell culture

1048

1049

1000

1040

More than 20 strains of vacuolating virus have been recovered in our laboratory. Table I shows the origin of 8 of the strains examined most extensively. The vacuolating virus has appeared as a contaminant in the seed stocks of all of types i through 7 adenovirus. These seeds had been prepared in cultures of rhesus or cynomolgus monkey kidney and were obtained from Dr. R. J. Huebner or were brought to this laboratory by one of us (M.R.H.) from Walter Reed Army Inst. of Research. Additionally, vacuolating virus was recovered from seed stocks of Myxovirus parainfluenza 1 and 3, from the SA virus, and from the respiratory syncitial agent received from Dr. R. Chanock and Dr. K. Habel.

Vacuolating virus strains $207, S211 and S215 were derived from types 1, 2 and 3, respectively, of Dr. Albert Sabin's live attenuated poliomyelitis vaccine. For recovery of vacuolating agent, the poliovirus pras neutralized by homologous polio immune serum prepared in rabbits immunized with Syverton's HeLa cell lines of poliovirus propagated in human stable amnion cell culture. The infectivity titer of the vacuolating virus in each of the Sabin vaccines ranged from 10-3.7 to 1045. These isolates of vacuolating virus were identified in serum neutralization tests with rabbit antisera against prototype strain

in such infected cells. Nuclei of infected cells may appear normal or disorganized internally. Limited fusion of the cytoplasm of adjoining infected cells may occur but, predominantly, each cell maintains its own integrity. Thus, the syncitium or giant cell formation, so characteristic of measles, foamy virus and certain other virus infections, is not prominent in vacuolating virus infection and this serves as a distinguishing feature. Terminal cytopathic changes occur in the cultures 5 to 10 days post-inoculation when the infected cells aggregate together and detach from the glass leaving behind only small islands of normal or degenerated cells. The cytopathic changes caused by vacuolating virus appear to be quite distinct from those described for Hull's 4 C.P.E. groups of simian viruses or for Malherbe's and Harwin's 7 simian agents.

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(S.V.2, 16, 18, 19) or by group 4 (S.V.3, 6. 20) antisera. In these tests, only a negative finding had significance since the antisera employed were prepared using virus grown in rhesus or cynomolgus monkey kidney cell cultures and these might also have contained vacuolating virus.

Host range. A preparation of vacuolating virus strain 776 which titered 10-5 in grivet kidney culture was tested in the various primary and line cell cultures shown in Table III. The virus was cytopathic for patas kidney and for rhesus monkey testicle, but these cells were about 30 to 300 times less sensitive than grivet kidney. Vervet monkey kidney has also been reported highly susceptible to the vacuolating virus(11). Grivet and vervet monkeys are separate races of the species Cercopithecus aethiops.

No definitive cytopathic change referrable to the vacuolating virus was observed in any of the remaining primary or line cell cultures, shown in the table, when observed for eight to twelve days. The virus persisted for at least 7 days in most of the cultures without increase in titer. Limited proliferation might have occurred. Titers as high as 108.5 have been obtained with vacuolating virus grown in grivet kidney.

The susceptibility of suckling mice and of embryonated hen's eggs to the vacuolating virus was tested. Suckling mice, inoculated intracerebrally, subcutaneously or intraperi

Strain 776 of vacuolating virus was not neutralized by antiserum against C.P.E. group 4 strain S.V.6, S.V.26 or S.V.29 antisera furnished by Dr. Hull. Additionally, it was not neutralized by Myxovirus parainfluenza 1 or 3 antisera or by such SA, S.V., or foamy virus antisera as were available to us. Vacuolating virus strains 776, 175, 953, 1095 and 584 were neutralized by pooled antisera against Hull's group 1B (S.V. 20, 23, 25, 27) and the last three strains by group 3 (S.V.12. 28, 59). None was neutralized by group 2

TABLE III. Titration for Cytopathic Effect of Vacuolating Virus Strain 776 in Various Cell Cul

tures.

Titer based on eytopathic end point, 8 days

10-4.5

104

testicle

10

kidney <10°

<10°

<100

Line cell cultures HeLa