Karl Habel

Resistance of polyoma virus immune animals to transplanted polyoma tumors.

Discussion and Summary By producing an inapparent infection in adult hamsters or C57 mice with a single dose of diluted polyoma virus these animals subsequently manifest a relative resistance to transplantation of isologous polyoma fibrosarcomas. These sarcomas, although originally induced by virus inoculated into newborn animals, no longer contain any demonstrable virus or viral antigens. This resistance can be overcome if a large enough number of tumor cells are given in the challenge transplant; it is apparently specific for tumors of polyoma origin and would appear to be cell mediated since hyperimmune polyoma antiserum is not capable of transferring such resistance to normal mice. The most logical explanation of this phenomenon is based on the hypothesis that polyoma induced tumors contain a cell antigen which is not present in normal isologous cells and the degree of this antigenic difference must be small. This new antigen would be produced by the normal cell after it has been transformed as the result of polyoma virus infection, both in the infected newborn and adult animal. The newborn being immunologically non-reactive would tolerate the foreign antigen and allow the transformed cells to multiply and produce a tumor. The adult animal being immunologically mature would reject the transformed cell with its foreign antigen and in so doing become hyper-reactive or resistant to the tumor when challenged with the transplant. This hypothesis is consistent with the findings of Law and Dawe(3) that adult mice given polyoma virus after whole body X-radiation do develop tumors. If this hypothesis can be proven by immunological experiments, it would suggest that the genome of the virus-transformed cell either now contains viral genome or has been genetically altered by a non-lytic virus infection.

SPECIFIC COMPLEMENT-FIXING ANTIGENS IN POLYOMA TUMORS AND TRANSFORMED CELLS.

Abstract One or more new specific antigens are present in “virus-free” polyoma tumors, as demonstrated by their complement fixation reaction with serum from animals carrying transplantable tumors. These antigens cannot be found in normal animal tissues or in tumors induced by other viruses or by methylcholanthrene. Mouse and hamster cell tissue cultures after exposure to polyoma virus are positive for the antigen and corresponding control cultures are negative. The presence of the antigen in these tissue culture cells is not related to the presence or absence of infectious virus in a carrier culture relationship or to the ability of the culture cells to produce tumors in isologous animals. Sera from tumor-bearing animals having high titers of CF antibody against tumor antigen are negative for antiviral antibody activity. Some sera from virus immunized animals are negative against the tumor antigen but have high anti-viral antibody titers. The presence of demonstrable anti-tumor CF antibodies is not necessary for virus-immunized animals to resist challenge with an isologous polyoma tumor.

Virus-cell relationship in a carrier culture of HeLa cells and Coxsackie A9 virus.

Abstract Although relatively resistant to infection with Coxsackie A9 virus of monkey kidney (MK) culture origin, HeLa cells have been found capable of supporting growth of this virus. When heavy inocula of the A9 virus were used in cultures of HeLa cells adapted to growth in medium containing 10% human, horse, or calf serum, the cultures showed viral cytopathogenic effects (CPE) but recovered after addition of fresh growth medium. These cultures could be passed serially for many cell generations with consistent recovery of the virus in the supernate. The term “carrier culture” has been used to designate this type of virus-cell relationship ( Lwoff, 1953 ). No evidence for a lysogenic state was found in this system. Animal or human immune serum eliminated virus from the cultures and clonal isolates of cells from the carrier culture were negative for virus. Only a small proportion of the cells in carrier cultures were infected. Prolonged cultivation of the carrier cultures tended to select not only cells with increased resistance to Coxsackie A9 infection, but also virus with altered properties. Virus from carrier cultures differed from the original virus grown in MK by its (a) lack of virulence for suckling mice, (b) increased virulence for HeLa cells, (c) slow plaque formation in MK plaque plates, and (d) antigenic composition. The evidence presented indicates that the principal factors responsible for continued maintenance of the carrier cultures are the relative insusceptibility of HeLa cells to Coxsackie A9 virus, especially in the state of cellular activity present in growth medium, and the prevention of high multiplicities of virus in the cultures by rapid thermal inactivation.

Relationship of polyoma virus and tumor in vivo.

Abstract The results of a serum protection test in newborn hamsters infected with polyoma virus indicated that tumor induction took place rapidly and directly. Protection against tumor induction occurred only if antibody was present at the time of virus inoculation. Even at that time, some animals developed subcutaneous sarcomas at the site of virus inoculation and most of these tumor-bearing animals did not produce hemagglutinin-inhibiting (HI) antibodies. Although the virus was necessary for tumor induction, direct evidence that it was required for tumor maintenance was lacking. A previously established transplantable hamster tumor in its thirteenth passage showed virus in the tumor and antibody in the host, whereas all other passages were negative. This transplant line was not induced to produce virus by in vivo treatment with X-ray, cortisone, or starvation, nor by X-ray and ultraviolet irradiation of tissue cultures made from the tumor. Two transplantable tumor lines were established in C57 BL mice from subcutaneous sarcomas developing as the result of virus inoculation of newborns. In one line, tumors were consistently negative for virus and donor adults negative for antibody. The other line was consistently positive for virus. Cells in tissue cultures of either line were susceptible to virus challenge.

The effect of interferon on SV40 T antigen production in SV40-transformed cells

Abstract Continuous passage in the presence of interferon failed to reduce the content of SV40 T antigen in a line of SV40-transformed mouse cells (3T3). However, marked inhibition of SV40 T antigen formation resulted when normal 3T3 cells were pretreated with interferon and subsequently infected with SV40 virus. The implications of the undiminished translation of viral genetic information in transformed cells in which an interferon-induced antiviral state exists are discussed. It is suggested that SV40-transformed cells produce molecules of messenger RNA (mRNA) containing both host and viral information, and that the presence of some host genetic information on the mRNA molecule renders it insensitive to the interferon system.

The Effect of Anticellular Sera on Virus Multiplication in Tissue Culture.

Abstract Viruses causing a cytopathogenic effect (CPE) in tissue cultures can be nonspecifically inhibited by anticellular sera. Not only CPE but actual multiplication of viruses is suppressed even when antisera are applied late in the growth cycle of some viruses. Different viruses vary in their ability to be inhibited and for certain viruses greater inhibition can be demonstrated in a cell system homologous for the anticellular serum. However, there is species crossing when antisera to human cells are tested in monkey kidney tissue culture. This phenomenon is due to an effect of the antisera on the cell and is not antiviral. The inhibitory activity can be removed by adsorption with red blood cells or the proper tissue cells. Limited evidence suggests that the antiserum action takes place at the surface of the cell or in the cytoplasm. Cross reactions in neutralization tests employing standard typing sera may be caused by this phenomenon.

Role of polyoma virus and interferon in a herpes simplex virus infection in vitro

Abstract A continuous line of mouse embryo cells persistently infected with polyoma virus (carrier culture 23-P) was observed to be resistant to challenge with herpes simplex virus. Resistance was dependent upon the input multiplicity of the herpes virus inoculum. At low multiplicities, the cytopathic effect (CPE) due to herpes virus infection was incomplete, regrowth of normal cells occurred, and a culture which elaborated both herpes and polyoma viruses (double carrier culture) was established. Elimination of polyoma virus by antibody treatment of the culture or cloning of the cells resulted in loss of resistance. The equilibrium between competing viruses and factors responsible for resistance was unstable, and three of the theoretically possible outcomes of such an equilibrium have been demonstrated: (1) the polyoma infection persisted while the herpes virus was eliminated from the culture; (2) the double carrier culture was destroyed in association with a rising titer of herpes virus; and (3) the double carrier state was maintained. Resistance of the original polyoma carrier culture and maintenance of the double carrier state appear to depend on the production of an interferon. Through the addition of exogenous interferon to susceptible, polyoma-free mouse cell cultures (3-B), the same three outcomes of herpes virus infection which had been observed in the resistant 23-P cultures could be reproduced. These results suggest that the resistance of the 23-P polyoma carrier culture to challenge with herpes virus is due to the additive effect of the interferon induced by both the polyoma and herpes infections, either of which alone is ineffective.

ISOLATION OF ENTERIC VIRUSES FROM CASES OF ASEPTIC MENINGITIS

In recent years there has been increasing evidence that the clinical syndrome of aseptic meningitis, including nonparalytic (NP) polio, may be caused by a number of etiological agents.I The etiological relationship existing among lymphocytic choriomeningitis, the arthropod-borne encephalitides, herpes, mumps, poliovirus, and leptospirae is now generally recognized. Prior to the development of tissue-culture methods for isolation of viral agents from large numbers of specimens, most of the evidence for etiological relationships had consisted in isolation of the virus from cases during the acute clinical illness and demonstration of increasing titers of specific antibodies during convalescence. I n some instances the isolation of the agent from spinal fluid or central nervous system (CNS) tissue a t autopsy and the production of the typical clinical and pathological picture in experimental animals have been strong arguments in establishing etiological relationship. Certain of the Coxsackie B viruses have been implicated by their frequent association with clinical disease and by isolation from spinal * However, no controlled epidemiological investigation of outbreaks of aseptic meningitis has been made where virus isolation rates in ill and control population groups could be compared. I n 1952, an investigation of the distribution of poliovirus types in the United States and Canada made available a number of virus isolates from cases of “nonparalytic In 1955, agents were isolated from both nonparalytic polio cases and from some clinically diagnosed as aseptic meningitis. The distribution of virus types isolated from these patients will be presented in this report. In the 1952 study, 125 nonparalytic polio patients were studied in the Children’s Hospital in Washington, D. C. Comparable specimens from 125 ward patients were also available in the same hospital, from 171 outpatient children a t the same hospital, and from 127 children and adults in a section of the community from which some of the ‘Lpolio’’ patients originated, all sampled a t the same time. From these groups the relative numbers of isolations of poliovirus, A and B Coxsackie, and certain ECHO viruses were determined.

Polyoma tumor antigen in cells transformed in vitro by polyoma virus

Abstract “Foreign” cellular antigens previously found in in vivo induced polyoma tumors of the hamster and mouse were sought in cells transformed in vitro by this virus. In the case of hamster embryo cells maintained in continuous culture for over a year since being transformed, the evidence for the presence of polyoma tumor antigens was equivocal. However, when a tumor produced by a recently transformed hamster embryo culture was used, tumor antigens were readily demonstrated. Mouse embryo cells transformed in vitro by virus, and a tumor derived from them, also contained the tumor antigens. The presence of the tumor antigens was determined by showing that polyoma virus-immune animals were resistant to tumor production after challenge with the in vitro transformed cells or with early transplant passage tumors derived from them. Resistance could be shown against a challenge with transformed mouse embryo culture cells carrying infectious virus, as well as against a transplantable tumor derived from them in which no virus could be demonstrated. No resistance could be shown in virus-immune mice challenged with a tumor derived from an uninfected control mouse embryo culture.

Sensitivity and resistance of type 1 polioviruses to an inhibitor in certain horse sera.

Abstract An inhibitor against type 1 poliovirus associated with the gamma globulin of certain horse sera was investigated. Addition of inhibitory horse serum (IHS) at a concentration of 2 1 2 % in the overlay medium caused marked reduction in plaque size and a delay of 24–48 hours in plaque development. Plaque counts were unaffected at this serum concentration, but were reduced by 50 % with 5 % serum. Inhibitor-resistant particles capable of forming normal-sized plaques in the presence of IHS were found in a plaque-purified pool of type 1 poliovirus (Mahoney strain). Based on plaque size, inhibitor-sensitive and -resistant particles could be differentiated in naturally occurring type 1 virus in stools of patients with paralytic poliomyelitis. The inhibitor had no effect on plaque development of type 2 and 3 polioviruses, Coxsackie A9, vaccinia, or encephalomyocarditis virus (EMC). The inhibitor differs from the previously described bovine serum inhibitor in not having neutralizing ability; no evidence was found that it is viral antibody. Growth-curve experiments showed that the inhibitor reduces rate of adsorption of virus to susceptible cells and delays release of virus from infected cells.