Search

A series of recombinant plasmids containing polyoma virus (PY) DNA were constructed, and their biological activity was evaluated in mice and in cultured mouse cells. While all of the recombinants studied contain the complete, potentially infectious viral DNA, in no case was the intact recombinant PY-plasmid DNA, or live Escherichia coli containing the recombinant plasmids, capable of inducing PY infection of mice, either by feeding or by parenteral injection.

Inoculation of suckling hamsters with 2 x 10(8) live cells of Escherichia coli K12 strain chi1776, carrying the complete genome of polyoma virus in a recombinant plasmid, failed to induce tumors in any of 32 recipients. Also, lambda phage DNA and particles with a monomeric insert of polyoma DNA did not induce tumors. Purified recombinant plasmid DNA, as well as phage particles and DNA containing a head-to-tail dimer of polyoma DNA, showed a low degree of oncogenicity, comparable to that of polyoma DNA prepared from mouse cells. These findings support the previous conclusions, based on infectivity assays in mice, that propagation of polyoma virus DNA as a component of recombinant DNA molecules in E. coli K12 reduces its biologic activity many orders of magnitude relative to the virus itself.

The tumorigenicity of DNA from polyoma virus after cleavage with a variety of restriction enzymes was evaluated in suckling hamsters. Cleavage with enzymes that interrupt the region of the genome coding for the large tumor (T) antigen of polyoma virus markedly enhanced the tumorigenicity above that observed with DNA I of the virus. Cell lines established in vitro from tumors induced by polyoma virions, polyoma virus DNA I, or polyoma virus DNA that had been cleaved with restriction endonucleases in the early region all contain the polyoma virus middle and small T antigens but not the large T antigen of polyoma virus is not required for maintenance of the transformed state and probably not for initiation of tumorigenesis by viral DNA.

The biological activity of recombinant phage and recombinant phage DNA containing monomeric or dimeric polyoma DNA inserts was examined in mice and cultured mouse cells. Recombinant preparations containing a single copy of viral DNA were invariably noninfectious; molecules containing a dimeric polyoma DNA insert were at least seven orders of magnitude less infectious than polyoma virions after parenteral inoculation. No infection was detected with any recombinant preparation after oral administration.

The biological activity of polyoma viral DNA was evaluated in mice and hamsters. Viral DNA administered parenterally is about 4 to 5 logs less efficient than polyoma virions in establishing infection in mice. Supercoiled viral DNA was infectious for mice after parenteral administration, giving mean infective doses of 10(-3) to 10(-4) microgram. However, animals fed microgram quantities of polyoma DNA I did not become infected. Linearization of viral DNA with R.EcoRI or R.BamHI, which are single-cut enzymes cleaving in the early and late regions of the genome, respectively, reduced the infectivity for mice approximately fivefold. Approximately 10% of newborn hamsters inoculated intraperitoneally with polyoma DNA I developed tumors. In contrast, the same amount of viral DNA which had been cleaved in the early region with R.EcoRI induced tumors in 50% of inoculated hamsters.

Mouse polyoma virus was stored at 4 degrees and -60 degrees C. for 8 weeks without any loss in hemagglutination or infectivity titer; storage at 37 degrees for 8 weeks reduced infectivity titer by approximately 2.5 log(10) units. Repeated freezing and thawing of infectious tissue culture fluid had no effect on virus titer. The stability of the virus to storage for 1 week at 4 degrees C. was unaffected by suspension of infected tissue culture fluid in saline, demineralized distilled water, or 5 per cent horse serum in Eagle's basal medium. Heating the virus for 30 minutes at 60 degrees C. had no effect on infectivity and hemagglutination titers. Heating at 65 degrees C. for 30 minutes produced a 3 log unit loss of infectivity and a 4-fold decline in HA titer, and heating at 70 degrees C. for 30 minutes usually produced complete inactivation of infectivity and HA. The virus was resistant to ultraviolet irradiation; ultraviolet irradiation for 2 hours caused a 4 log unit decrease in infectivity titer without affecting the HA titer. The virus was resistant to exposure to 2 per cent phenol and 50 per cent ethyl alcohol but was inactivated by 100 per cent ethyl alcohol, and ethyl alcohol-iodine mixtures. Lyophilization had no effect on the stability of the virus.

The relationship of polyoma (parotid tumor) virus infection to spontaneous and x-ray induced lymphocytic neoplasms (leukemia) in mice has been studied in two sublines of the high leukemic AKR strain, in the high leukemic C58 strain and in the x-ray responsive C3Hf/Bi strain. There was found to be no correlation of hemagglutination-inhibiting (HI) antibody with leukemia appearing at various times throughout life, and there was no evidence of increased risk of developing leukemia in mice with prior antibody to polyoma virus. Virus isolation experiments accomplished with AKR leukemic mice showed that HI antibody status of these mice had the same significance with regard to presence or absence of detectable virus as was previously observed for non-leukemic mice from infected colonies. The results of this study were interpreted as supporting the concept that the repeated association of polyoma virus and experimental transmission of leukemia is fortuitous.

Eight mouse colonies were surveyed for prevalence of antibody to mouse polyoma virus. Frequency of HI antibody varied from 0 to 84 per cent in adult mice in different colonies. Antibody was infrequent in mice less than 3 months of age, and increased in frequency with age. There was no evidence that infection was specific for particular mouse strains. The highest frequency of infection was found in colonies in which breeding mice are housed in proximity to mice inoculated with polyoma virus or passage tumors, and within an infected colony, the incidence of infection was greatest in rooms housing mice inoculated with polyoma virus. Mice from a colony free of antibody became infected when held in room or cage contact with virus-inoculated mice, but at very low rates except in mothers of inoculated litters. These results were interpreted as indicating that artificial contamination of the environment is an important factor in determining the prevalence of infection in the colonies observed. There was no correlation between polyoma infection and spontaneous leukemia in AK. mice.

THREE PROCEDURES HAVE BEEN COMPARED FOR USEFULNESS IN TITRATION AND DETECTION OF POLYOMA VIRUS: production of cytopathic effect (CPE) in mouse embryo tissue culture, production of HI antibody after inoculation into weanling mice (MAP test), and production of tumors in suckling hamsters during a 3 to 5 week observation period. The tissue culture and mouse antibody production tests were generally comparable in sensitivity, reproducibility, and time required to obtain results. Titration by tumor production in suckling hamsters was not suitable for quantitation because of marked variation in susceptibility among animals. Virus was detected in tissues of normal mice from spontaneously infected colonies by either production of CPE in mouse embryo tissue culture or by the MAP test; virus was found in organs of 15 (58 per cent) of 26 mice with antibody, and 2 (8 per cent) of 24 mice without antibody.

Treatment of guinea pig erythrocytes with types A and B influenza viruses rendered them inagglutinable by polyoma virus; also, the inhibitory effect of ovomucin on polyoma virus hemagglutination was destroyed by pretreatment of the ovomucin with various myxoviruses. These results indicate that polyoma virus and myxovirus erythrocyte receptor sites are identical. However, no destruction by polyoma virus of its own or of myxovirus receptors or inhibitors was detected. No serologic relationship was detected between polyoma virus and members of the myxovirus group; differences in size and stability further indicate their distinctness. No evidence was found of biologic or serologic relationship of polyoma virus with encephalomyocarditis virus or mouse encephalomyelitis virus.