ABSTRACT

STUDIES ON ind VIRAL ETIOLOGY OE VERRTCA VULGARIS
IN TISSUE CmLL CLLlURE

By Hajime hayashi

Clinical observations and early human injection experiments have
shown that verruca vulgaris (common human wart) was the result of
infection by a virus. Although many attempts have been made, the agent
has not been recovered in tissue cell cultures.

Over 200 samples of verruca tissues from 195 patients were tested
in 13 different tissue cell lines. Only when cells from verruca tissue
were placed in contact with the Ab cell cultures was cytopathic effect
observed. Cell-free material from verruca tissues or cell cultures
failed to produce visible cytopathic effect in vitro. Subcultures of
three strains of the agent from three different patients were maintained
through 23, 26, and 32 serial passages, showing that the agent could
be prOpagated by cell to cell transfer. Gamma globulin and homologous
sera, when used undiluted in a cell-serum neutralization test prevented
cell to cell transfer of the agent. Dilution of the sera or globulin
did not prevent infection indicating that large amounts of antibodies
were necessary to prevent the agent from infecting normal cells.

hany attempts were made to establish a new epithelial cell line
from normal human skin and verruca tissues but none was successful.

Because of the hazards involved in injecting humans, an attempt
was made to establish in animals the agent isolated in tissue cell

cultures. Nonkeys, rabbits, hamsters, mice, and chickens were injected

suuulas ON qua VIRAL EIIOLOGY OF vaRRUQA VULGARI3
IN TISJUE CELL UULlURE

By

Ha j ime Haya shi

A THfiSIS

submitted to
Michigan State Lniversity
in partial fulfillment of the requirements
for the degree of

DOOlOR OF PHILOSOPHY

—Department of Eicrobiology and Public Health

1961

This work is
respectfully dedicated
to

IVE FIANCEE AND PARENTS

ii

‘."'."TT.‘ '1, 77 '3
Avh-4\)..4.hh-l um uni J

lhe autaor wisxes to exoress his sincere appreciation to
Or. Ialter H. flack, without whose constant interest, patient
understanding, and willinjness to help, this investigation would

not have been possitle.

Appreciation is extended to Dr. T. L. Lallnann, Or. a. D.
Devereux, Dr. J. J. Stockton, Jr. J. L. Eairley, and Dr. R. L.
Anderson for their helpful sugjestions and criticisms of this

thesis.

lhanks are also due to the American Cancer
financial assistance enabled the author to hold
graduate research assistant.

Sincere thanks are exoressed to hiss Joyce

Brs. Iail D. Riegle, hrs. Janet E. lussell, and
for their cooperation and technical assistance.

iii

Society whose
the position of

A. Remsberg,
other workers

TABLE OF CONTde

INTRODUCTION . . . . . . . . . .

I. Perspectives in viral etiology of tumors

II. The etiology of verruca vulgaris

remains mm E~ETHODS . . . . .

I. Tissue cell culture studies .

II. Tissue cell culture neutralization tests

III.

IV. Electron microscope observations of

seen in verruca tissues .
DISCUSSION . . . . . . . . . . .
ADDENDUM . . . . . . . . . . . .
SLHJARY . . . . . . . . . . . .

BIBLIOGRAPJY . . . . . . . . . .

iv

U)

the particles

Attempts to infect laboratory animals with verruca tissue
and wart agent recovered in tissue cell cultures

PAGE

11
15
2b
2b
he

51

61

67

68
69

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Wable

Table

Table

Table

Table

l.

2.

3.

h.

S.

6.

7.

8.

9.

10.

ll.

12.

13.

1}.

LIST OE TAELES

Origin of cell cultures used . . .

Tests on extracts of frozen warts from

62 patients in two tissue cell lines . . . . . . . . . .

The results of subculturing (blind passage) nutrient

fluids from human conjunctiva cell cultures

Tests on fresh wart tissues from 6h patients
in eight tissue cell lines

The results of subculturing (blind passage) nutrient

fluids from unfrozen wart extracts in two tissue cell

lines

The results of subculturing (blind passage) tissue

and nutrient fluid from unfrozen wart fragment . .

Growth in primary cell cultures using
fresh fragments of wart tissues

Initial and subcultures of wart agent from three

patients in AU 08].]. CUlturBS o o o o o o o o o o 0

cells

Attempts to cultivate the wart agent in other epithe-

lial cells after isolation in the Ab cell cultures

Attempts to grow AU cells in medium

Survey of agent present in wart tissues
from 69 infected individuals

containing animal sera . . . . . . . . . .

The effect of 35 or 37 C incubation temperature
on the ability of cells or cell-free passage material

I O

to infect AU and monkey kidney cell cultures . . . . . .

serum

Results of neutralization tests employing mixtures

Results of neutral'zation tests employing mixtures of
infected AU cells and gamma globulin or homologous

of infected Ab cells and rabbit anti-infected AU cells,

anti—normal AU cells and anti-infected cell-free
nutrient

15.

Results of inoculating mice with fresh verruca

tissue extracts, fragments and tissue cell cultures. . .

25

27

28

28

32

38

1:7

50

52

\t “1 .
Pauli:

Table 16. Day-old chicks inoculated with AD infected
C811 SUI‘CUltureS o o o o a o o o o o o O 0 o o o o o O 0 59

Table 17. Inoculation of adult hamsters with infected
and non-infected AU cells . . . . . . . . . . . . . . . 60

Table 18. Inoculation of suckling hamsters with normal
and infected AU cells . . . . . . . . . . . . . . . . . 62

Table 19. Results of injecting rabbits with normal AU cells
anCi imji‘ected 0/111) cells 0 O O O O O O O O O O O O O O O O O 62

l.

2.

h.
S.
6.

7.

8.

Implant of wart fragment . . . . . . . . . . .

Fibroblast-like growth from wart implant . . .

Early stages of cytopathic effect of

seeded with fragments of wart tissue .

Cytopathic effect on AU cells

Cytopathic effect, end results

Normal AU cells monolayers . . . . .

AU cells

Cytopathic changes of wart tissue implant

and AU cell culture . . . . . .

Zone of cytopathic change between wart tissue

implant below, and AU cells above

vii

PA GE:
31
31

35
35
36
36

L2

112

INTRODUCTION

The theory that viruses might cause tumors was originally proposed
by Borrel (1903). Although his attempts to discover the cause of
tumors were unsuccessful, he suspected that tumors were the result of
viruses infecting the cells. The discovery of the viruses of leukosis
by Ellermann and Bang (1908) and sarcoma by Rous (1911) stimulated a
search for tumor-inducing viruses. These initial studies were very
fruitful for several years and much of the fundamental knowledge we
have today, regarding virus-induced tumors, came from this early work.

Recently Dmochowski (1961) summarized that within the past ten
years 150 viruses have been isolated, many of which have been found
responsible for various types of tumors in animals.

Although many viruses in animals have been isolated as causative
agents of tumors, no proven tumor-producing agent of man has been iso-
lated. Many agents have been isolated from man, eSpecially from leuke-
mia, but it is not known if these agents are the cause of leukemia.

Man is not a satisfactory'experimental animal.

The present literature states that the cause of human warts (verruca
vulgaris) is a filterable agent. This stems from the very early work of
Ciuffo (1907) who ground up wart tissue, filtered the extract and ino-
culated himself with the filtrate. Many attempts have been made since
1907 to cultivate the agent of verruca but none were successful.

The experiences in this thesis were those dealing with the many
attempts to isolate an etiological agent from human wart tissues. The
methods employed were many but centered around cell cultures, the newest

tool for the study of viruses. Experimental animals were inoculated by

many routes, employing known "tricks of the trade". Tissue extracts
and fluid phase nutrients from cell cultures were studied under the
electron microscope in the search for this agent.

Any agent isolated from.wart tissues would have great value as a
model antigen. The agent would open the door to many unknown questions

about virus-induced tumors and especially the virus-tissue relationship.

I. Perspectives in viral etiology of tumors

 

A neoplasm is an independent overgrowth of tissue which serves no
useful purpose and is usually destructive to normal tissues. The term
tumor is properly applied to any neoplasm, benign or malignant.

Malignant tumors are those that endanger life. They infiltrate and
destroy the tissue locally and metastasize to distant tissues. The malig-
nant cells are undifferentiated or embryonic. Benign tumors are composed
of well-differentiated tissues which approach or attain the structure of
adult tissues. They do not spread widely throughout the body, grow so
rapidly, or kill so regularly as malignant tumors. The word cancer is
used in a popular sense to designate any malignant tumor.

Methods of investigating tumor-inducing viruses do not differ from
those used in the study of other infective agents. These same biological
and immunological techniques have been used since the beginning of this
century.

When an infectious process was studied and the etiological agent was
not observed under the microscope, the infection was thereafter considered
as a virus infection. The inoculation of animals with this infectious
material sometimes resulted in the transmission of the infection and
serial transfers were accomplished. The discovery of various types of

filters that would retain bacteria and permit smaller agents to pass

through into the filtrate soon demonstrated viruses.

Ciuffo (1907) reported that the Berkefeld N filtrate of wart material
from man was infectious and demonstrated the long interval between the
time of injection and the appearance of warts on his own arm. In the
following year Ellermann and Bang (1908) observed that when blood, emul-
sions of organs or filtrates of organs from leukemic chickens were inocu-
lated into healthy birds, fowl leukemia developed in the experimental
birds. Rous (1911) discovered the transmission of sarcoma in chickens by
injecting cell-free filtrates into experimental chickens. The following
year Rous et a1. (1912) demonstrated that osteochondro-sarcoma was a virus—
induced disease of birds. Claude and Murphy (1933) reviewed the reports
of virus-induced tumors of chickens and included myxosarcoma isolated by
Eujinami and Inamoto in 1910, the spindle-cell sarcoma by Lange in 19lh,
the fibromyxosarcoma by Hayashi in 1915 and the endothelioma by Begg
in 1927.

The successful isolations of agents from chickens prompted a search
for the causative agent in tumors from other animals. Oral papilloma-
tosis of dogs has the characteristics of warts in man. De Monbreun and
Goodpasteur (1932) removed the warts from a dog and, after extracting
the tissue, filtered the fluid phase through a Berkefeld filter. 'With
this cell-free filtrate they were able to produce warts in puppies
but ndsin rabbits, rats, mice, guinea pigs, kittens nor monkeys.

Shops (1932a) upon examining a cottontail rabbit, discovered a
fibroma-like tumor. After the tumor had been minced and extracted with
saline solution, rabbits were injected with the extracty'and tumors were
produced. Shops (1932b) later used cell-free extracts to reproduce this

tumor in rabbits and demonstrated the relationship of the fibroma and

myxomatosis agents of rabbits.

One of the many peculiarities of host susceptibility was demonst-
rated by Rous and Beard (1935). They Observed that, when rabbit papil-
loma virus was introduced into wild cottontail rabbits, the tumors grew
but many regressed with time. In the cottontail rabbits the disease was
a benign infection. When the tumor material was removed from the cotton-
tail rabbits and the virus extracted from the tissues and then injected
into domestic rabbits, the result was malignant infection.

Bittner (1936) was the first to demonstrate that carcinoma tissue
contained a transmissible agent. The agent was isolated from the mammary
glands of mice, and filtrates from infected tissues readily infected
experimentally injected mice. It was also shown that infection took place
naturally between mother and offspring through the milk. Isolation of the
Bittner mammary virus led to the speculation that in man cancer infection
takes place in new born and only manifests itself in later life. This
theory has not been proved for man, nor under these circumstances can we
directly compare man and mouse. Support of this theory was strenghthened
by the isolation of "mouse poliovirus" or Theiler's virus (1937) in mouse
colonies. The agent was a latent virus frequently found in experimental
mouse colonies. Although the virus was latent in most of the individuals,
approximately one per cent of the animals developed paralysis and died
from the infection. The virus was transmitted to the young through contact
with adults in the colony. Theiler's virus was demonstrated as a latent
infection by applying certain physiological insults to adult members of
the colony.

Mouse leukemia, a virus-induced disease, was discovered by Gross
(1951). His success was dependent upon injecting cell-free material from

leukemic AK mice into newly born inbred 03H mice. The results of Gross

have been confirmed by several investigators (Furth et al., 1956; Stewart,
1953; Woolley and Small, 1956). Friend (1957a, b) independently observed
leukemia in mice and was able to snow transmission to new born mice by
injecting cell-free splenic extracts. This leukemia virus resulted in
myelogenous leukemia while the virus isolated by Gross most often produced
lymphocytic leukemia.

In an attempt to apply the knowledge learned through the study of
mouse leukemias, tissue extracts from.hunan cases of leukemia have been
injected into new born mice. Leukemia has resulted in a small proportion
of the injected animals, and only certain strains of mice appear to be
susceptible to the agent. The type of leukemia developing in the serial
passages can not be predicted.

Certain undisputable facts have resulted in the study of transmissible
virus-tumors. l) The younger the experimental animals used, the greater
the possibility of isolation success. 2) Most virus-induced tumors in
animals require a much longer "incubation period" than has been eXperi-
enced with the usual infective-type virus diseases. 3) The resulting
tumor produced by injecting a certain tumor-inducing virus can not be
predicted. Since tumors are classified by what they look like to the
pathologist, the name given to a piece of tumor tissue varies.

The fact remains that viruses are repeatedly recovered from cell-free
extracts of tumors from animals and these extracts can produce disease when
introduced into the susceptible experimental animal.

The in'vitro cultivation of tumor-producing viruses has been accom-
_plished repeatedly for the past thirty years. It was not until recently

that tissue cell cultures were extensively used to propagate these agents.

Chicken sarcoma virus was cultivated ig‘zitzg by Furth and Stubbs
(l93h). They placed pieces of chick tissue in plasma clot cultures and,
after outgrowth of cells, seeded the cultures with chicken sarcoma virus.
In this manner they were able to show multiplication of the virus in the
cultures. The plasma clot culture method was used extensively in the
cultivation of viruses during this period.

The EB 33252 cultivation of all types of viruses expanded when
Dulbecco and Vogt (l95h) discovered that when tissue cells were treated
with trypsin and then placed in cultures, a monolayer of cells developed.
The cell sheet could be then seeded with virus and the resulting virus
multiplication be visually observed by plaque formation.

Evidence of virus multiplication was called the cytopathic effect
(OPE) and was applied to tumor inducing virus by Lo et al. (1955). They
observed that fragments of normal chicken fibroblasts, when grown in
plasma clots, were lysed after seeding the culture with Rous sarcoma
virus. The OPE consisted of degeneration of cells and lysis of the clot.
Serial subcultures of the nutrient fluid from these cultures produced
degeneration of cells, giant cell formation and a variety of cell shapes
and sizes. Non-infected control cultures remained uniform.

Mouse leukemia virus was successfully cultivated in monkey kidney
monolayer cells by Stewart et al. (1957). The OPE seen in these cultures
was not typical as the cells of the culture appeared to pile one adjacent
to another. However, fluid from the cultures, when injected into mice,
produced leukemia. When mouse embryo cells were used for cultures, Eddy
et al. (1958b) found the mouse leukemia agent produced a cytopathic effect.
In early passages of the agent the cells did not appear different from

those in control cultures but in two or three weeks some differences in

cell morphology did take place. As "blind" passages were continued cyto-
pathic changes included an increase in pyknotic cells until the majority
of the cells in the cultures were affected, causing removal of the cells
from the wall of the tubes. Finally adaption of the agent to the ig
32232 culture resulted in subcultures all of which produced cytopathic
effect. The agent induced leukemia when injected into mice.

The agent of visceral lymphomatosis was shown to produce cytopathic
effect in cell cultures of chicken embryo liver by Sharpless et al. (1958).
Fontes et al. (1958) also described cytOpathic effect with this agent
cultivated 23.31259 on chicken cells. In both reports, CPE was observed
only after several "blind" passages in cultures. The cytopathic effects
were characterized by Fontes et al. as refractable granules appearing in
the cytOplasm of the polygonal epithelial-like cells; the nucleus became
pyknotic and this was followed by shrinkage of the cytoplasm.or rounding
of the cells. Some cultures contained groups of cells which formed grape-
like clusters. The cells finally detached from the tube wall.

Stewart (1953) described the induction of a parotid gland tumor in
mice. The mice had been inoculated, within a few hours after birth, with
a cell-free extract of tissue from a spontaneous leukemia in a mouse.

The methods Stewart used were those described by Gross (1951) for produc-
ing leukemia in mice. Stewart's results indicated that the virus she
recovered had oncogenic activity and was unique in several prOperties.

The agent called the polyoma virus, has stimulated search for other agents
producing tumors in animal and man, and has been extensively used as a
model system. The agent and its resulting tumor have the following pro-
perties:

l) The tumor inducing agent can be propagated i2 vitro in tissue

cell cultures (Stewart et al., 1957; Eddy et al., 1958a; Stewart et al.,
1958).

2) Its oncogenic activity not only crosses strain barriers in mice
(Stewart et al., 1958; Mirand et al., 1958) but also crosses species
barriers producing tumors in hamsters (Eddy et al., 1958a; Stewart et
al., 1960) and rats (Eddy et al., 1959).

3) It has the properties of many of the usual types of infectious
viruses such as high antigenicity (Stewart and Eddy, 1959a; Stewart et
al., 1959), production of cytopathic changes in cell cultures (Eddy et
al., 1958b) and hemagglutination of erythrocytes from several animal
species (Eddy et al., 1958c). The hemagglutination reaction provides
a simple method for the titration of viral activity and specific anti-
bodies in sera.

As with many of the tumor-inducing agents, polyoma virus when in-
jected into animals produces more than one type of primary tumor. That
is why the agent has been called the polyoma virus (Eddy et al., 1958b).
The agent produces malignant tumors and has,in many respects, altered the
present concept of what is and what is not malignancy.

The serology of tumor resistance has a complex and unmeaningful
background. Animals surviving the tumor infection are resistant to
the agent. Serological demonstration of this resistance has presented
certain difficulties. Burmester (1955) has shown passive resistance in
chickens 33.21252 and ig 3213 with the lymphomatosis agent. Sharpless
et al. (1958) and Fontes et a1. (1958) demonstrated serum neutralization
against the lymphomatosis agent ig 21333. The antibodies were produced
by repeated injection of lymphomatosis virus into chickens. Neutralizing

antibodies to the Friend's leukemia agent of mice have been reported by

Friend (1957a, b).

The polyoma virus probably has much to offer as a model in attempt-
ing to understand the reaction of host resistance to the tumor agents.
This agent is without doubt the most active of the tumor agents with
regards to serological tests and antibody formation. Neutralization,
hemagglutination inhibition and complement fixation tests can be applied
to the polyoma agent and its specific antiserum (Eddy et al., 19580;

Rowe et al., 1958). On the other hand, several agents produce antibodies
to tumor cells as well as the agent (Beard et al., 1957; Cheever and
Janeway, l9hl; Eckert et al., 19553 Gorer and Law, 19h9; Kabat and Furth,
l9hl; Law and Malmgren, 1951 and Malmgren et al., 1951).

Other means of detecting tumor-inducing agents have shown some
success. Electron micrographs of ultra-thin sections of tissues or
ultracentrifuge concentrates of tissue extracts have resulted in objects
which appear to be virus particles in these preparations (Dmochowski,
1959 and 1961). Visual observations of these virus-like particles from
tumors or tissues and confirmation that they are the causative agent are
lacking. Electron micrOSCOpy has its painful limitations when used alone,
but when combined with other means of exploration it becomes a valuable
asset to the whole understanding of the problems.

The presence of tumor-inducing agents, although poorly understood
because of many complicating factors involved, has been proved in many
ways. Not all of the viruses producing tumors have been reviewed in
this work, however, the following general outline lists those animal
tumors which are thought to be produced by viruses.

(1) Avian leukosis complex

a. Lymphomatosis

b. Myeloblastosis

10

c. Erythroblastosis
(2) Avian sarcoma
(3) Amphibian renal carcinoma
(h) Mammalian papillomatosis
a. In man
b. In dogs
c. In rabbits
d. In cattle etc.
(5) Mouse mammary carcinoma
(6) Mouse leukemia
a. Lymphocytic
b. Myelogenous
Recently Stewart and Irwin (1960) described proliferative changes in
certain cell cultures inoculated with five specimens taken from patients
having tumors. The first was a surgical specimen from a papillomatosis
growth of the tongue in a 73-year-old male. The second specimen was from
tissues of a 22-month-old child with an embryonal tumor. The next was
urine from a 6-year-old boy with a renal neuroblastoma and the last Speci-
men was from an 8-year-old child with Hodgkin's disease. mxtracts from
all of these specimens were placed in contact with cell cultures of human
tissues and "blind" passages were made. It was noted that after 3—h pass-
ages over h-5 months, focal areas with abnormal migration of the cells
were observed in the inoculated cultures. The cultures were transferred.
Although proliferative effect was reproducible in tissue cultures by
serial passage of the culture fluids, it appeared that cells or cell
particles were necessary for transfer, and culture activity was lost by

filtration or centrifugation of the tissue culture fluid. Transfer of

11

cells instead of nutrient fluid is new to tissue culture prepagation of
viruses.

'Heller et al. (1958) were able to prOpagate varicella and herpes
zoster in tissue cultures only if infected tissues were placed in contact
with the cultured cells. Passage also required transmission of in-
fected cells from culture to culture.

Rowe et al. (1957) was able to prepagate the human salivary gland
virus in cultures of human skin only when infected tissue was used as
inoculum and transfer material contained infected cells. It appears
that in some cases the parasite is closely associated with the cell and

infection takes place 12 vitro from infected cell to "normal" cells.

II. The etiology of verruca vulgaris

 

Verrucae, or common human warts, are classified (Anderson, 1957)
as benign tumors, representing a thickening or projection of epidermis.
Several adjectives are used to describe verrucae depending upon their
shape, location or the clinical features of the lesion. Verruca vulgaris,
plantaris, digitala, filiformis, juvenilis and senilis are all warts and
probably are all caused by a common etiological agent.

Verruca vulgaris is most commonly seen on children and is found on
fingers, hands, arms and legs or generally those areas of the body not
clothed. They occur singly or in groups. Histologically, verruca vul-
garis is characterized by a papillary acanthosis surmounted by friable
keratic material. The cells of the stratum granulosum are often acidophilic
and vacuolated. A loose infiltration of various mononuclear cells may
be present in the papillae (Anderson, 1957).

The infective nature of warts has no dated record. People knew that

12

warts could be spread by contact and history has many amusing stories
about this strange condition. The cause and cure of warts also have

much history in the development of man. In ancient times as well as
today, persons were identified by their warts. The exact cause of warts
has interested many people but the first recorded experiments regarding
their etiology was in 1896 when Jadassohn (1896) published his work,
beginning in 189b, on the cause and transmission of warts. Jadassohn
removed wart tissue from his patients and after grinding up the tissue

in salt solution or by using small fragments of tissue, injected the skin
of his own arms and hands with the Specimen. His work extended to experi-
ments on colleagues, and 7h inoculations resulted in 33 reproductions of
warts. Jadassohn's experiments settled the question regarding the
infectious nature of the wart, but he made no attempt to explain the
etiological factor involved. In 1907, Ciuffo (1907) produced warts by
injecting Berkefeld N filtrates of extracted wart tissue. He inoculated
the patient from whom the wart was originally removed and the skin of

his own hand. After 5 months incubation period, warts appeared in both
subjects. Not only did Ciuffo confirm Jadassohn's original observations
but he demonstrated that a filtrate of wart material was infectious and
that tissue cells and parasites were excluded from the inoculum. Ciuffo's
work has been repeatedly referred to regarding the viral etiology of
human warts. The filterability of the wart agent was confirmed by

Serra in 1908. In 1919 Nile and Kingery (1919) filtered the extracts

of warts and reproduced warts with these filtrates. Kingery (1921)
produced a second generation by removing an experimentally produced wart
and after filtering the extract, reinoculated a volunteer, producing

again a wart growth. In all of these transmission experiments in human

13

beings, a prolonged incubation period of 6 months or more was observed
before the appearance of the wart growth. In most of the experiments,
the warts regressed shortly after their appearance.

The cause of warts was not discussed again until 1953 when Bivins
(1953), a poultry pathologist, removed a wart from.his finger and
seeded an extract of the wart upon the chorioallantoic membrane of a
developing chick embryo. Resulting plaques on the membrane were misinter-
preted by Eivins to be the result of wart virus. Siegel (1956) found
that the agent isolated by Bivins was a strain of avian pox virus.

Strauss et a1. (19h9, 1950) examined concentrated extracts of wart
tissues under the electron microsc0pe. They found crystalline particles
which they considered to be virus particles in many of their preparations.
The particles in the crystalline array averaged 52 mu in diameter but
averaged 68 mu when not observed in the crystalline—like clusters. The
observations of Strauss et a1. were not confirmed by Siegel (1960). The
latter used a refined concentration method and found a large variety of
particle sizes, but some preparations contained a 16 mu virus-like par-
ticle which Siegel considered more uniform and suggested might be the
wart agent.

The etiological agent of human warts has been considered to be
virus since 1907 when Ciuffo filtered the wart tissue extract and repro-
duced warts by injecting the filtrate into the skin of his hand. The
viruses producing warts in dogs (De Monbreun and Goodpasture, 1932),
cattle (Olson et al., 1959) and deer (ShOpe et al., 1958) have been
characterized to some extent because eXperimental animals were available.

The results presented here are efforts to isolate the human wart agent

by tissue culture methods.

lb

Throughout this thesis the word ”agent" has been used synonymous
with "virus" although the wart agent as described herein does not fit

the classical criterion of a virus.

MATERIALS AND I-‘lE'l‘HODS

Many of the reagents and composition of the reagents were altered
during the three years these experiments were conducted. Whenever
standard or stock reagents proved satisfactory their use was continued
but many times the compositions were changed to meet the needs of the
work in progress. The various solutions of growth and maintenance
media are given here as stock solutions and when altered the changes will
be given in the results of that particular set of eXperiments. All
reagents used were diluted with water which had been freshly distilled
through three cycles in an all glass distilling apparatus.

Tissue cell cultures were obtained, whenever possible, from commer-
cial sources. When not commercially available, the cultures were obtained
from the best source available, generally the author describing a certain
cell line. In the latter case, the culture was sent to the laboratory
by air and thereafter the culture was prOpagated in the laboratory. In

table 1, the cell lines used and their sources are listed.

Reagents
1. Hanks' balanced salt solution (ass, Hanks and Wallace, 191,19)

 

Solution 1: Dissolve l.h g Ca012 in 200 ml distilled water.

Solution 2: Dissolve the following in 900 ml of distilled water.

Glucose ........ 10 grams KH2 .......... 0.6 gram
bIaCl . O O C C . O O O O O 80 " Na 21-{P0h0 2H2O . O C . O. 6 "
K01 . O O . . O I O C . . . 1L " Phenol red O C O O . . O . 2 "

MgSOh.7H20 ..... 2 "
The two solutions were separately autoclaved at 10 lbs pressure
(115 C) for 10 min. The solutions were mixed together and approximately

0.35 g NaH003 was added to adjust the pH to 7.2.

15

l6

 

 

 

 

Table 1: Origin of cell cultures used.
Medium Cells Obtained

Name Tissue Origin Required From

Human Normal human* Chang BME** & Tuskegee Inst.,

conjunctiva conjunctiva 10% H.S.x Alabama

AU Normal human“ I'Theeler et al. was or Yh‘lvfm‘L ldaeeler, Univ. of
skin & 20% H.S. Virginia, Va.

HuS 3075 Normal hunan* Perry et al. were 109 & Natl. Cancer Inst.,
skin 10% A.S.xx NIH & Tissue Bank,

Natl. Med. Center

Human Jejunum of* Henle BNE & Microbiol. Assoc.,

intestine human embryo 10% H.S. Bethesda, Md.

Human Human adult* Girardi BEE & Microbiol. Assoc.,

heart heart 10% H.S. Bethesda, Md.

HeLa Carcinoma of* Gey BEE & fiicrobiol. Assoc.,
human cervix 10% A.S. Bethesda, Med.

T-l Human kidney & van der Veen 199** & Pitman Moore Co.,
lung reticulo- % A.S. Zionsville, Ind.
sarcoma

Monkey Epithelium, 199 & Microbiol. Assoc.,

kidney primary culture 5% A.S. Bethesda, Md.

MAF Human embryonic BEE & Microbiol. Assoc.,
skin and muscle 10% H.S. Bethesda, Md.

D—189 "Malignant trans-6) mm & Microbiol. Assoc.,
formation of Leighton 10% H. S. Bethesda, Md.
human foreskin"

Detroit-98 Human adult* Berman and mm a Microbiol. Assoc.,
sternal marrow Stulberg 10% H.S. Bethesda, Md.

Human Epithelium, BEE & Payne, Univ. of

amnion primary culture 10% H.S. Mich., Mich.

Chick liver Embryonic chick 199 & Dept. of Nicrobiol.,
liver 5% A.S. iich. State Univ.

* Cell lines propagated in this laboratory.

** See code for abbreviations under reagents.

x H.S. u

xx A.S. a

Human serum.

Animal serum.

2.

(100x) and 1 m1 of amino acid mixture (100x) were added.

17

Basal medium, Eagle (BI-IE, Eagle, 1955)

 

To 98 ml of Hanks' balanced salt solution, 1 m1 of vitamin mixture

amino acid mixtures consisted of the following chemicals and each was.

dissolved in 1 liter of distilled water:

3.

Vitamin mixture

 

Biotin ............... 0.1
Choline .............. 0.1
Folic acid ........... 0.1
Nicotinamide ......... 0.1
Pantothenic acid ..... 0.1
Pyridoxal ............ 0.1
Thiamin .............. 0.1
Riboflavin ........... 0.01
i—Inositol.2H20 ...... 0.18

Amino acid mixture

 

Arginine .............
Cystine ..............
Histidine ............
Isoleucine ...........
Leucine ..............
Iymme.u.u.u.u.u
Methionine ...........
Phenylalanine ........
memhm u.u.n.n.
Tryptophan ...........
Tyrosine .............
Valine ...............
Glutamine ............

Yeast extract medium (YEN, Robertson et al., 1955)

 

0.
3:333:01)

ormt‘rmmmmmooMI—J

ONI—‘ONHONMNOl—‘N

3

-u.-‘-.
‘---‘-

To Hanks' balanced salt solution add 0.1 per cent yeast extract

(Difco) and 0.35 per cent glucose.

h.

Mixture 199 (Morgan et al., 1950)

 

This medium.was obtained as a commercially prepared reagent and

consisted of the following chemicals dissolved in 1 liter of distilled

water:

DL-Tryptophane o o o o o o .o o o o 20 mg

DL-Phenylalanine ........ 50
DL-Isoleucine ........... hO
DL-Leucine ..............120
DL—Methionine ........... 3O
DL-Serine ............... 50
DLPThreonine ............ 6O
DL-Valine ............... 50
DIPOLAlanine ............ 50
L-Proline ............... hO
L-Hydroxyproline ........ 10
Aminoacetic acid ........ 5O
LaArgine ................ 70

II
II
fl
3‘
I!
ll

L-Histidine .........
L-Lysine ............
Sodium acetate ......
DL-ASpartic acid ....
Dbflbmmflcadd..n
L-Tyrosine .........
L—Cystine ...........
Adenine HCl .........
Guanine HCl .........
Cysteine HCl ........
Xanthine ............
Hypoxanthine ........
Thymine .............

20
70
so
60
150
ho
20
lo

0.30

10

0.30
0.30
0.30

The vitamin and

II
II
I!
I!
I!

of the

urBCil 00000000000000000 0030
Vitamin.A 0000000000000010

Calciferol .............
Cholesterol ............
I'lerladion ......OOOOOOOOO

Disodium Ot-too opherol

phOSphate ...........
Niacine ................
Niacinamide ............
Pyridoxine H01 .........
Pyridoxal H01 ..........
flumunHm.u.u.u.n.
Riboflavin .............
Calcium pantothenate ...
L-Inositol .............
p-Amindbenzoic acid ....
Choline chloride .......
D-Biotin ...............

0.10
0.20
0.01

0.01

0.01

18

mg

I!
II
II
H

0.025"
0.025"
0.025"
0.025"
0.010"
0.010"
0.010"
0.050"
0.050"
0.500"

"

Folic acid ............ 0.01 mg
L-Ascorbic acid ....... 5.0 "
Ghmflmume.u.u.u..‘&0 "
D-Ribose .............. 0.50 "
Adenylic acid ......... 0.20 "
Adenosine triphOSphate. 0.010 "
L-Glutamine ...........100 "
Ferric nitrate ........ 0.10 "
mml..u.n.u.u.u MED "
KCl ................. boo "
CaCl ............... 1h0 "
IagS .7H20 0000000000 100 I!
Mg012 6H0 .......... 100 "
NazHPOh. SHgO ........ 60 "
M%H%..u.u.n.n. &) "
Mumme.n.u.n.u.lmm "
Phenol red .......... 20 "

The final pH was adjusted to 7.2 with NaHC03 just prior to use.

NCTC 109 (MoQuilkin et al., 1957)

Purchased from Microbiological Assoc., Bethesda, Md. and consisted

following chemicals dissolved in 1 liter of distilled water:

L-Alanine 000000000000 0031-148 mg

L-ot-Amino-n-b utyric

acid ..........
L-Arginine ...........
L-Asparagine .........
L-Aspartic acid ......
L-Cystine ............
D-Gluoosamine . . . . . . . .
L-Glutamic acid ......

0.0551
0.2576
0.0809
0.0991
0.1009
0.0320
0.0826

L-Glutamine .......... 1.3573

Glycine ..............
L-Histidine .........;
Hydroxth-Proline ....
L-Isoleucine .........
L-Leucine ............
L-Lysine .............
L-Methionine .........
L-Ornithine ..........
L—Phenylalanine . . .. ..
L—Proline ............
L-Serine .............
L-Taurine ............

L-Threonine

L-Tryptophan 000000000
L-TerSine 00000000000
L'Valine 0000000000000

0.1351

‘0.l973

0.0h09
0.180h
0.20hh
0.3075
0.0hhh
0.0738
0.1653
0.0613
0.1075
0.0h18
0.1893
0.1750
0.160h
0.2500

N
I!
I!
N
I!
I!
I!
n
I!
H
II

Thiamin H01 ............
Riboflavin .............
Rfiimmhmlml..n.u..
Pyridoxal HCl ..........
Niacin .................
Niacinamide ............
Calcium pantothenate ...
Biotin .................
Folic acid ....
Choline chloride .......
i-Inositol .............
p-Amindbenzoic acid ....
Cyanooobalmin ..........

Vitamin A

calCiferOI 0000000000000
Menadion ...............

o<-Toc opherol phosphate .

Qumufimm..u.u.u..

Ascorbic acid ..........

Cysteine HCl ...........
DiphOSphopyridine ......
nucleotide .......
TriphOSphopyridine
nucleotide .......
Coenzyme A .............
Cooarboxylase ..........

0.00025 mg
0.00025 "
0.000625"
0.000625"
0.000625"
0.000625"
0.00025 "
0.00025 "
0.00025 "
0.0125 "
0.00125 "
0.00125 "
0.1 "
0.0025 "
0.0025 "
0.00025 "
0.00025 "
0.1 "
0.5 n
2.6 "
0.070 "
0.010 "

0.025 "
0.010 "

l9

Flavin adenine Sodium acetate ..... 0.5 mg
dinuoleotide ....... 0.010 mg N801 ............... 68.0 "
tridine triphOSphate .. 0.010 " KC ................ 0.0 "
Deoxyadenosine ........ 0.1 " Ca012 .............. 2.0 "
Deoxycytidine H01 ..... 0.1 " MgSOb.7H20 ......... 1.0 "
Deoxyguanosine ........ 0.1 " NangPOh ............ l.h "
Thymidine ............. 0.1 " MaHCO ............. 22.0 "
5-Lethylcytosine ...... 0.001 " Dextrose ........... 10.0 "
Glucuronolactone ...... 0.018 " Tween 80 ........... 0.125 "
Sodium gluouronate .... 0.018 " Phenol red ......... 0.2 "

The final pH was already adjusted.
5. Tryosin solution

Trypsin was used to separate cells from tissue or removing cells
from the walls of tissue culture tubes. The concentration of trypsin used
varied with its use. Usually a concentration of 0.25 g of trypsin (1:250,
Difco) in 100 ml calcium free Hanks' solution was used (See solution 2 above
in Hanks' balanced salt solution). The trypsin solution was sterilized
by Seitz filtration. A l per cent solution was used to separate cells from
tissues.

6. Antibiotic solution

 

Penicillin and streptomycin were incorporated into all nutrient
fluids used for tissue cultures. Usually 100 units of penicillin and 100
ug of streptomycin per ml of nutrient fluid were employed. This concentra-
tion was increased when heavily contaminated tissues were encountered.

7. Chick embryo extract

 

Fertile chicken eggs were incubated for 9 days. Only those embryos
appearing live and well developed upon candling were used. The embryos
were removed from the egg, washed in B38 and transferred to the barrel
of a 20 cc syringe fitted with a circular stainless steel screen.

Pressure was applied to the plunger of the syringe forcing the tissue out

of the barrel and into an equal volume of B38. The extract was then cen—

20

trifuged at 2500 r.p.m. for 20 minutes, placed in tubes and stored at

-20 C until use. Just prior to use the tube was quickly thawed and the
fluid again centrifuged at 2500 r.p.m. to remove any formed sediment.

The supernatant fluid was used to form serum clots or as nutrient for cell

cultures.

Tissues

wart tissues were removed by total enucleation (Ulbrich et al., 1957).
Tissues removed at the dermatologists' offices were placed in sterile
vials and either refrigerated at h C or frozen in freezing compartments
of refrigerators. For shipment to the laboratory the vials were placed
in contact with dry ice.

Specimens of warts were also removed in the laboratory. The tissues
removed were placed in Hanks' balanced salt solution containing anti-
biotics and held at h C for 2h-h8 hours. When needed normal skin was
removed. Depending upon their use, the specimens were prepared in
various ways. Cell-free extracts were prepared by grinding the wart
tissues in a mortar with a pestle using alundum as abrasive. Minimum
amounts (5 per cent wart suspension) of basal salt solution containing
antibiotics, were used as diluent. The suspensions were centrifuged in a
horizontal position at 1500 r.p.m. for 10 minutes to remove the cells and
debris. Fragments of wart tissues were prepared by mincing the tissue as
fine as possible with a blade after the tissue had been stored overnight
in Hanks' balanced salt solution and antibiotics. Trypsinized wart tissues
were prepared by placing the tissue in one per cent trypsin solution at
h C for several hours. The cells were separated from the trypsin solu-

tion by slow speed centrifugation.

21

Sterility tests

 

Sterility control of all tissue culture naterial was accomplished
by seeding sample Specimens into several screw capped tubes containing
brain heart infusion broth (Difco). The tubes were incubated at 37 C
and examined each 2h hours for bacterial growth. Tissue samples were
considered free from.bacteria only if no growth occurred in the broth
after 72 hours incubation. Occasionally, tissue cell cultures were
tested for the pleurOpneumonia-like organisms by seeding samples of cell

cultures, including cells, into Special pleuropneumonia medium (Difco).

Tissue other than wart tissue

 

Many attempts were made to establish a tissue cell line from normal
skin. Normal human skin was obtained on demand from John Dunkel, M. D.,
pathologist, Edward Sparrow Hospital, Lansing, Michigan. Foreskins from
new born infants were collected daily. Skin from adults was obtained
upon demand at the hospital or removed from volunteers in the laboratory.
In eXperiments using human normal skin, the skin was placed in Hanks'
balanced salt solution containing antibiotics and stored at h C for 2h
hours or until the sterility control tests indicated bacteria were not

present in the specimen.

Blood and blood products

 

Blood serum was used to supplement nutrient solutions for tissue
cultures. Sera from various animals were used depending upon the type
of cell cultures and whether the cultures were being propagated or main-
tained. Human blood was obtained through Joseph Venier, M. D., Lansing Red
Cross Blood Bank. Whole blood was collected from volunteers, the clot

was allowed to form over night and the cells were separated from the

22

serum by centrifugation. Strict sterility tests were made by placing
0.1 ml of each serum sample into multiple tubes of brain heart infusion
broth. Any sample found contaminated was discarded.

Blood was drawn from horses or calves when needed and the serum and
cells separated as described for human blood.

Fractionated human blood samples were supplied by the Michigan

department of health laboratories, Lansing, Nichigan.

Experimental animals

 

White Swiss mice, Nebster's virus susceptible, were reared in the
laboratory. Pregnant females were observed daily for birth of their
young. Litters were sometimes pooled to obtain sufficient numbers of
animals. Adult hamsters were purchased and laboratory bred. Upon birth
of the young, the day old hamsters were used in the experiments. Chickens
used were hatched from eggs in the laboratory. After hatching, chicks
were removed from the incubator, inoculated and placed in heated cages.
Half-grown white laboratory rabbits were injected with wart tissues,
tissue culture fluids and cells. No attempt was made to use day old

rabbits. The monkeys used were Macaca gynamolgus and purchased from

 

animal dealers. The ages of the monkeys were unknown but all had deciduous
teeth. X-ray "tanning" of the skin was accomplished by irradiation of
the abdomen at 50 cm in 6 doses at h8 hour intervals until 1000 r. had

been accumulated.

Electron microscope observations

 

Extracts were made from.wart tissues and normal and infected cell
cultures. The method has been described by Strauss et al. (l9h9, 1950).

The extracted material, after slow speed centrifugation to remove debris,

23

was centrifuged at 6000 r.p.m. for h5 to 60 minutes in a refrigerated
International Multispeed centrifuge. The sediment was resuspended in
small amounts of distilled water. Minute amounts of the resuspended
sediment were placed upon electron microscope collodion prepared screens

and after drying were shadow-casted with tungsten oxide before examination.

RESULTS

I. Tissue cell culture studies

 

A. Inoculation of wart material into tissue cell cultures

 

The early attempts to isolate the wart agent were made by standard
virus-tissue culture methods. The frozen wart tissue was extracted by
grinding the tissues in a mortar and diluting the pulp with basal salt
solution. After centrifugation to remove the debris, the supernatant
fluid was then placed in contact with monolayer tissue cell cultures.
Observations were made daily of each culture and any morphology altera-
tions recorded. Any change in cell morphology of any culture was followed
by serial "blind" subcultures using the nutrient fluid as passage material.
All subcultures were observed for as long as the cells could be maintained.
Occasionally morphology changes in cells were seen but consistent results
were lacking upon subcultures. In table 2 the results are given for 78
samples of frozen wart samples from 62 different patients.

Two epithelial cell lines were used and the results showed no evi-
dence that the agent was affecting the cells. subcultures of the nutri-
ent from the human conjunctiva cultures were made at 5 to 1h day intervals
(table 3). Seven samples were subcultured twice; one sample was passed
h times and two samples were passed 5 times but no evidence of agent
activity was observed.

Evidence that the causative agent of warts would withstand freezing
was lacking, therefore, arrangements were made to have wart tissue removed
in the laboratory. Fresh wart material was extracted, treated with trypsin
or separated into small fragments prior to placing in contact with the

different tissue culture cell lines.

2h

25

Table 2: Tests on extracts of frozen warts from 62 patients
in two tissue cell lines.

 

 

 

Treatment of No. Days
Cell Line No. Samples Fart Tissue Observed
Human
conjunctiva 66 Extracts 13—21
human embryonic
skin (was) 12 Extracts 18-2L

 

 

Total: 78

 

Table 3: The resmlts of subculturing (blind passage) nutrient
fluids from human conjunctiva cell cultures

 

 

 

Cell No. do. Passage No. Days
Line Samples Passages Line Observed Results
Human 7 2 human lB-lh per Negative
conjunctiva conjunctiva passage

1 h d man l3-lh per Hegative

conjunctiva passage

2 5 human 13—1h per Negative
conjunctiva passage

 

26

The results of testing 75 samples of unfrozen wart tissue in eight
different cell lines are presented in table h. All cell cultures were
observed for as long as they could be maintained but no evidence of viral
activity was observed. In two of these tests (Detroit—98 and D-189 cell
lines) the fresh tissue was first extracted and the resulting cell-free
extract was frozen. This was necessary because of the difficulty in
obtaining the two cell lines at the same time that fresh wart tissues were
available. The results given in table h represent the wart tissues
from 6h patients. In two cell lines, T-l (human kidney and lung reticulo-
sarcoma) and HuS 3075 (human normal skin), "blind" subcultures were made.
The cell free nutrient fluid from previous cultures was used as inoculum.
As indicated in table 5, no evidence of agent growth was observed in any
of the cultures of any passages. In table 6 the results are given of
a "blind" subculturing of combined tissue culture cells in nutrient fluid.
The original cultures were inoculated with small (1 mm3) fragments of
wart tissue. After 1h days the cells were mechanically removed from the
tube wall and suspended in the nutrient fluid. Both cells and fluid
constituted inoculum for subsequent passages. As can be seen, five
samples were subcultured three times and observations indicated that
there was no visual evidence of agent activity.

In viewing the results given with the various types of tissue cultures
used, it was obvious that the agent in frozen or fresh wart material was
not easily adapted to 22.223E2 cultivation and a search was made for

other cell cultures.

B. Attempts to cultivate primary human skin

 

As there was no evidence of growth of the wart agent in the epithelial

cell cultures, attempts were made to establish a culture of human skin.

27

Table h: Tests on fresh wart tissues from 6h patients

in eight tissue cell lines.

 

 

 

 

 

 

 

 

 

 

 

Cell No. Samples Treatment of Ho. Days
Lines Tested Fart Tissue Observed
Human
conjunctiva 8 Extracts 13-21
Human embryonic l Trypsinized cells 1h
skin (RAF) 7 Extracts 1h-21
nunan heart 1 Trypsinized cells 1h

12 Extracts 1L-2l
human intestine l Trypsinized cells 21

10 Extracts 21
Detroit-98 (Human Extracted and
sternal marrow) 10 then frozen 1h
D—189 (Kalignant
transformation of 10 Extracted and 11
human foreskin) then frozen
T-l (Human kidney
and reticulosar- 2 Pieces 21
coma of lung) 2 Extracts 21
an: 3075 (duman 6 Pieces 111
normal skin) 5 Extracts lb

 

 

Total:

79

 

28

Table 5: The results of subculturing (blind passage) nutrient fluids
from unfrozen wart extracts in two tissue cell lines

 

Cell No. No. Passage No. Days
Lines Samples Passages Line Observed Results

 

T—l (human

kidney & 2 2 T-l 1h Negative
reticulo-

sarcoma of

lung

 

Hus 3075
(human normal 5 3 Hus 3075 lb Negative
skin)

 

Table 6: The results of subculturing (blind passage) tissue cells
and nutrient fluid from unfrozen wart fragment.

 

 

 

Cell No. No. Passage No. Days

Lines Samples Passages Line Observed Results
HuS 3075

(human normal 5 3 HuS 3075 lb per Negative

skin) passage

 

29

The methods used to prepare the skin for cultures were a modification of
those described by Perry et al. (1956) and Wheeler et al. (1957). The
skin sample was placed into Hanks' balanced salt solution containing anti-
biotics to free the tissue of viable bacteria. This was followed by
permitting the tissue to remain in a one per cent trypsin solution until
soft and then transferring to Eagle's basal medium containing five per
cent chicken embryo extract and 20 per cent inactivated human serum.

After standing for 10-18 hours at h—S C, the dermis and epidermis could

be separated. The former was then placed into fresh growth medium and
shaken to disperse the cells. Horizontal tube cultures were prepared
containing approximately 100,000 cells per ml. Eagle's medium with chicken
embryo extract and human serum was used for growth medium.

Twenty-four times skin from different individuals was tested in an
attempt to establish a skin culture. None of the attempts were success-
ful. The growth obtained was usually fibrdblast-like in nature and only
occasionally was a monolayer of cells observed. Those cultures surviving
two weeks sometimes contained epithelial-like cells and were subcultured.
Cultures transferred by trypsinization or mechanical dispersion of the

cells failed to grow.

C. Cultivation of wart tissue cells

 

All attempts, so far, to isolate the causative agent of warts in
tissue cell cultures had been fruitless. Attention was then given to the
epithelial cells of wart tissues. Attempts were made with 9 different
samples to cultivate trypsinized wart cells by the methods described for
normal human skin. Only fresh wart tissues were used but the enzyme

treatment apparently destroyed the cells.

30

Small fragments (l mm3) of wart tissues were treated with antibiotics
in balanced salt solution and then transferred to culture tubes. To
attach the tissue fragments to the tube wall, the tissues were allowed
to dry slightly prior to the addition of nutrient fluid. The nutrient
used was to per cent human serum and 5 per cent chicken embryo extract
in Hanks' balanced salt solution. The cultures were observed daily.
Because of the mass of cells, the nutrients required changing each 2h
hours.

The cultures at 2h hours showed an outgrowth of epithelial cells
surrounding each tissue fragment but by'h8 hours the epithelial cells
stopped growing and were released from the tube wall. Figure 1 shows
a fragment of tissue with the cells growing out from the edge of the
tissue. At this time, fibroblasts were seen to grow out from the tissue
in long strands of cells. Figure 2 shows the fibroblast-like sheet of
cells after lb days growth.

In table 7 the results of ten attempts to grow fragments of tissue
are presented. The tissues used were from ten different patients. All
attempts resulted in growth of fibroblasts which could be maintained for
four weeks, at which time degeneration of the cells was observed. Two
cultures from two patients were subcultured (M.F. and L. J.). In the
former the subculture was observed to grow for three weeks and in the
latter the subculture survived for two weeks. It is doubtful if such
cultures would be of value, providing continuous cell lines were maintained,
as only fibroblasts were observed in the cultures after the first 2h-h8
hours of growth. Since verruca is primarily a tumor of epithelial cells,
it would seem reasonable that epithelial cells would be the most suscepti-

ble host for the agent.

31

 

Figure 1. Implant of wart fragment (h days,
unstained, x200).

 

Figure 2. Fibroblast-like growth from wart implant
after 1h days, unstained, x200 .

32

Table 7: Growth in primary cell cultures using fresh fragments
of wart tissues.

 

 

 

Initial Culture Duration No. of
Patient Cells in Veeks Subcultures

c. P. + * 2 N.D.'H
1‘4. F. 1 2 3
c. H. + h 11.1).
K. L. + 3 N.D.
P. E. + h W.D.
J. 1.. + h 11.1).
1. J. + h N.D.
L. J. + 2 2
r. o. + h LD.
J. I. + L '.D.

 

* * = Fibroblast growth.

we N.D. = Not done.

33

D. Cultivation of an agent in AU tissue cells

 

The history of the AU tissue cell culture has been described by
bheeler et al. (1957). The original cells came from a 17—year-old boy
(LL) and were established as a cell line for the study of virus infection
of the skin by theeler et al. The number of subcultures of this strain
of cells was not known and no attempt has been made to record these data
since the cell culture was received in this laboratorv. The cells are
epithelial and morphologically indistinguishable from the deLa neOplastic
cells of Gey (Jay at al., 1952). The nutrient requirement of the AU cells
is complex but satisfactory growth takes place in a medium consisting of
80 per cent yeast extract medium with 20 per cent inactivated human serum
as recommended by Vbeeler. Later, it was determined that Eagle's basal
medium could be substituted for the yeast extract medium. To maintain
the cultures two per cent inactivated calf serum was used. Both growth
and maintenance media contained antibiotics.

The AU cell line appears to be difficult to maintain as compared to
well established cell lines. The human serum used has a great deal to
do with the rate of growth as well as the length of time cultures can be
maintained without spontaneous degeneration of the cell cultures. Serum
from some individuals was found to destroy the cells.

The first attempts to cultivate the wart agent in AU cells were made
with fresh wart tissues. Patients with multiple wart infections were
chosen to provide ample wart material. Cell—free extracts and fragments
(l mm3) from each patient were prepared and each was seeded into three
tubes of AU cell cultures. The cultures were incubated at 37 C in the
horizontal, stationary position and observed each 2h hours for morphologi-
cal chanves in the cells. Uninoculated cell cultures were observed as

:1

controls.

3b

In those cultures receiving cell-free extracts of wart tissues, no
changes were observed. Those cultures receiving fragments of wart tissues
were found to be altered and, depending upon the amount of inoculum used,
the cells in some of the cultures were detached from the tube wall.
Although sterility control tests did not indicate the presence of bacteria,
it was at first considered that the tissue cell cultures were contaminated.
The procedure was repeated. This time fewer fragments of wart tissue per
tube were used as inoculum and the cultures were observed several times
during the first h8 hours of incubation. Again the AU cultures receiving
cell-free inocula were indistinguishable from the cells in the control
cultures. The cultures receiving wart tissue fragments were seen to change
from their normal morphology to a rounded cell which aggregated and finally
became separated from the cell sheet and tube wall. This cytopathic effect
(CPE) is illustrated in figures 3, h, and 5, and the normal cell sheet
of the AU cells is shown in figure 6. Cultures showing cytopathic effect
were subcultured in two ways. After the majority of the cells had become
detached from the tube wall, the nutrient fluid and cells from each tube
were pooled per specimen. The fluid was placed in centrifuge tubes and
spun at slow Speed in a horizontal centrifuge. The cell-free supernatant
fluid was separated from the cells. Only enough fluid was left on the
sedimented cells to act as a vehicle. The cell-free supernatant fluid
was used as inoculum for fresh cultures of AU cells. The cell suspension
(0.1 ml of heavy suspension) was also inoculated into fresh cultures.

Both sets of cultures were incubated at 37 C and observed repeatedly. As
in the original cultures, the cell-free inoculum produced no visible signs
of cytopathic effect. Although the time between inoculation and effect

was increased by several days, those cultures receiving the cellular

35

 

Figure 3. Early stages of cytOpathic effect of
AU cells seeded with fragments of wart
tissue. (Unstained, x2h0).

 

Figure 11. Cytopathic effect on AU cells.
(Unstained, x2110).

36

 

Figure 5. Cytopathic effect, end results.
(Unstained, x2h0 .

 

Figure 6. Normal AU cells monolayers.
(Unstained, x2h0).

37

inocula again were found to exhibit cytopathic effect and the cells were
detached from the wall of the tubes. Although the cultures receiving
cell-free inocula showed no effect and were indistinguishable from the
control cells, "blind" passages were made using the cell-free nutrient
fluids as inocula. No evidence of cytopathic effect was seen in these
series of cultures.

In table 8 typical results are given of the initial and secondary
passages of wart-infected cellular inoculum on the AU cell cultures.
Patient 1 (Hood), age 26, had a history of repeated wart infection on the
skin of both hands. Patient 2 (Oppelt), age 28, also had had warts removed
repeatedly. Patient 3 (Johnson), age 10, had extensive wart infections
on the skin beneath the distal margin of thumbs and index fingers. The
latter patient's infection was so extensive that the removal of additional
tissues would produce disfiguration. The patient was considered extremely
susceptible to infection by the wart agent.

To make certain that the wart agent was multiplying in the AU cell
cultures, repeated serial passages were made using 0.1 ml of a heavy
suspension of infected cells as inoculum. In table 8 the data are
presented for three strains of the wart agent that have been serially
passed 32, 26, and 23 times from patients, Hood, Oppelt, and Johnson
respectively. The nutrient fluids were changed every second day in the
serial passages and,considering the dilution and number of passages, there
was little doubt that the agent was prepagated from infected to uninfected
cells.

In an attempt to understand the mechanism involved in this unusual
circumstance, experiments were designed to disprove the results. Normal

fragments of skin (1 mm3) from parents of the patients from whom warts

38

owmmmmm ppmw

1_J
mo mm .m<

w m c
Q 35 E
u -

N
a. o mmm «Na

 

Amhmp NHV

Amhmp mv

.1 J
o om oa

 

Aconcaosv m pcmwpmm

.mwmwmmm some :H popmfldoocfi moLdp ....... Hm .... o¢ **

.poowmo cagpmmopho mcfizozm onduHSo Hams mammwp mo moans *

mwmmmmm spew

 

m m
* m * ¢
_ Amsma my
Nwm *N<
_ Amhmp av
*Hm *Ha
\fi Amuse me
W m J
0 O O
o * m * a

 

Apammmov N pamwpmm

ommmmmm pawn

b----

 

 

 

a 1.
*mo *mm *m< omwmmmm phage
\—; Amhmp m v
q ‘ la
*mo *wm *N< mummmmm uncomm
Ammmp OHV
*Hm *H¢ ommmmmm pmpfim
fl Amhmp.av
011 0. NJ mpamsm mph
m o * m * a sum: no aowpmfi

:soosH Hmflfimwno

 

Aeoomv H pamwpmm

.mopgpfido HHmo 34.:H mpcoflpmm conga
Scam pcowm paws Ho monspdsonwm paw HprHcH "w oflnma

39

were removed, normal skin from patients from whom warts had been removed,
foreskin from newborn and normal skin from laboratory personnel were used
as inoculum on AU cell cultures. CytOpathic effect was not observed
when any of the normal skin tissue was used as inoculum. Normal—appear-
ing, uninoculated AU cells were removed from the tube wall by physical
means and seeded onto monolayers of AU cell cultures. These cultures
were incubated and observed similarly to infected cultures but no cyto-
pathic effects were produced in the cell cultures. To eliminate the
possibility that wart tissues contained antibiotic resistant microorga-
nisms not present in normal skin, repeated cultures were made of the
inoculum-cell material to Special culture media for bacteria. Special
consideration was given and tests were made for the pleuropneumonia-like
organisms. No cultivatable bacteria were recovered from these control
tests. As will be seen later on, infected cells from serial passages
were injected intracerebrally into animals without producing meningitis,
a good test for the presence of bacteria.

The AU cells appeared to be the only cells showing evidence of
infection from contact with wart tissue. To determine if other epithe-
lial cell cultures could be infected withzhibcted AU cells, six epi-
thelial cell lines were tested. Table 9 represents the epithelial
cell lines used. The AU cells used as inocula were subcultures of the
agent using the patient's name to identify the strain. The infected
AU cell cultures were divided into cell suspensions and cell-free nutrient
fluids. Both samples were then used to inoculate cultures of the indi-
cated (table 9) epithelial cell line.

Although different strains were used and different passages of the

same strain, there was no evidence that the agent could be visually

hO

Table 9:

Attempts to cultivate the wart agent in other epithelial

cells after isolation in the AU cell cultures.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

AU Subculture Cell Line Observation
rPassage gflujtnres Inocula Period in Days
Hood 1, 13, 1h, 15* Human embryonic Cell-free 1h-21
Oppelt lo, 12 skin (HAF) nutrient

Johnson 8, 10, 12

Anderson 1

Dallman l

Sussex 5

Crail 2

Hood 1, 13, IE, 15’ Human embryonic Cell

Oppelt 10, 12 skin (MAE) suspension lh-2l
Johnson 8, 10, 12

Anderson 1

Dallman l

Sussex 5

Grail 2

Hood 15, 18 Monkey kidney Cell-free

Oppelt 12, 1h nutrient 21
Johnson 12, 13

Hood 15, 18 Monkey kidney Cell

Oppelt 12, 1h suspension 21
Johnson 12, 13

Hood 13 Human kidney and Cell-free

Oppelt lO reticulosarcoma nutrient 21
Johnson 10 of lung(T-l)

Hood 13 Human kidney and Cell

Oppelt lO reticulosarcoma suspension 21
Johnson 10 of lung (T-l)

Hood 28 HeLa Cell-free

Oppelt 21 nutrient 1h
Johnson 21

Hood 28 heLa Cell

Oppelt 21 suSpension 1h
Johnson 21

Hood 20 Embryonic Cell-free '
Oppelt 16 chick liver nutrient 1h
Johnson 15

Hood 20 Embryonic Cell

Oppelt 16 chick liver suSpension 1h
Johnson 15

Hunter, original Human amnion Cell

Anderson, original (primary culture) suspension 21
Porter, original

Johnson, original Human amnion Cell-free

Larkins, original (primary culture) nutrient 21

 

* Number following strain indicates passage number.

bl

identified in any of the epithelial cell lines used. Control evidence
of continued growth of infected AU cells is not presented in table 9.
but, in all cases, the subsequent passage of the agent was maintained

in AU cell cultures.

E. Mixed culture of wart and AU cells

 

To visualize what was happening when wart cells came in contact
with normal AU cells, small fragments (l mm3) of fresh wart tissue were
attached to the tube wall by the plasma clot method. After attachment
of the implants, nutrient fluid was added to the tube and the culture
was incubated horizontally at 37 C for 1h days. Observation of the
implants showed that fibroblasts were growing out from the edge of the
implants as illustrated in figures 1 and 2.

Normal AU cells, after removal from.the tube wall by trypsin, were
suspended in nutrient fluid at 100,000 cells per ml concentration. One
ml of the normal AU cell suspension was then placed into each tube
containing wart tissue implants. The AU cells were permitted to attach
to the walls of the tube by horizontal incubation at 37 C. The tubes
were placed so that the wart implants and AU cells were in the same
plane. After 2h hours incubation the cultures were observed repeatedly
to determine the results.

At the end of 2h hours incubation period, a zone of cytopathic effect
was seen around the wart tissue implants. The zone consisting of rounded
cells, continued to increase. By the fourth and fifth days the AU cells
and wart tissue implants had degenerated and the cells were detached
from the tube walls. Figures 7 and 8 show the zone of cytOpathic effect.

The results verify the fact that the wart agent present in the

implants was capable of infecting the AU cells and producing a progressive

A2

 

Figure 7. Cytopathic changes of wart tissue
implant and AU cell culture (Unstained,
x200).

 

Figure 8. Zone of cytopathic change between
wart tissue implant below, and AU cells
above (Unstained, x200).

h3

cytOpathic effect. As the reaction started at the periphery of the
implant and spread outward, it was considered that the agent in the
wart implant diffused outwardly from infected to normal cell. The
results observed were identical to plaque formation as seen in monolayers
of cells infected with other viruses. In this case, the agent was
present in wart tissue cells and not detectable in the fluid phase as
is customary in plaque formation with other viruses. The infection was
progressive as in the growth of virus forming a plaque. From the
experiment it could not be determined whether the fibroblasts seen in
the cultures were capable of infecting the AU cells or not. Although
the predominant cells growing out from the implant were fibroblasts,
occasional epithelial cells were present. The final result was

removal of all cells from the wall of the tube.

a

B. Survey of agent in wart tissues from infected patients

 

When it was found that an agent that infected AU cell cultures
was present in wart tissue, the next question to be answered was whether
this agent could be repeatedly isolated from.wart tissues.

warts from patients were removed in the laboratory and the tissues
were permitted to stand at h C in balanced salt solution containing anti-
biotics for 2h hours. Following the standing period, small fragments of
tissue (1 mm3) were removed from the wart tissue. Three tubes of AU
monolayer cell cultures were seeded with tissues from each patient. The
cultures were incubated at 37 C and observed daily for cellular changes.
No attempts were made to subculture those cultures showing typical
cytopathic effect. A decision as to whether the agent had been isolated
was difficult to determine. For want of more information an isolation

was considered when two or more of three tubes from the same specimen,

Lb

showed a marked cytopathic effect. Using this arbitrary criterion for
an isolation the data were compiled in table 10. From 21 patients (30.h3£)

the agent was recovered. The average ace of these patients was 22 years.

a
In h8 patients (69.57%) the agent coxld not be recovered, yet the negative
results came from a younger average age group than did the positive
samples. These data gave results just the opposite of what we eXpected

and will be discussed later. It must be noted that only 69 patients

made up the total number of individuals tested.

G. Attempts to use animal serum replacing human serum for the growth

 

of AU cell cultures

 

Proof that the agent isolated was the causative agent of warts
depended upon whether it would produce verruca wlen introduced into or
onto the skin of man. As the AU cell line required human serum for its
growth ant, as hunan sera may contain the jaundice virus, attempts were
made to adapt the AU cells to a medium containing animal serum. It was
also suSpected that hunan serum might contain antibodies to the wart
agent. If the latter were true, this might eXplain why virus was not
found in the fluid phase of the cultures. Animal sera were available
while human sera required additional time and arrangements.

Honolayer sheets of AU cells were prepared in standard tissue
culture tu as using yeast extract medium plus 20 per cent human serum
as nutrient. The cells were trypsinized by the standard aethod and
placed in tubes for attachment and the growth phase. The nutrient fluid
was yeast extract medium: only the serum concentration and origin was
altered. The serum composition of the first passage was 75 per cent

human serum and 25 per cent calf or horse serum (see table 11). All

tubes were incubated at 37 C. in a horizontal position. then the cells

LS

Table 10: Survey of agent present in wart tissues
from 69 infected individuals

 

 

 

 

 

Average age Location of Tissue
No . of Individuals
Patients Results (inyears) hand Foot Arm Leg, Unknown
21 Positive 22 23 l 1
he Negative 19 38 l 2 6 b
Total: 69 61 2 3 6 h

 

Table 11: Attempts to grow AU cells in medium
containing animal sera

 

 

 

 

Type No. Subculture Growth

Serum Attempts lst 2nd 3rd hth 5th 6th
Calf 3 +++ +++ ¢9+ ++ e -
Calf 1 ‘-++ +4 a. -

Calf 2 44+ + _

Horse 2 44+ ++ -

Human (control) 1 a4; ‘44 +44 s++ 44‘ as;

 

* 444 mxcellent, as Good, 4 Fair, - No growth.

to

had formed a monolayer sheet, the cells were subcultured. The second
passage nutrient fluid contained half human and half calf or horse serum.
The subcultures were continued until the cells were grown in nutrient
fluid containing no human serum.

The adaptive procedure above was followed by subculturing the cell
line in the type of serum to which it had been adapted, i.e. calf or
horse sera. In table 11 the results of these experiments show that only
a single additional passage could be made when other than human serum
was used in the nutrient fluids for the cells. By the second or third
subculturing the cells failed to multiply in the provided nutrient.
Although a total of eight attempts were made with sera from three calves
and a horse, the results showed that human serum was an essential compo-

nent in the nutrient of the AU cell cultures.

H. The effect of temperature of incubation upon bound and fluid-

 

phase verruca agent

 

Kilham (1959) reported that when fibroma and myxoma viruses were
grown in tissue cultures at 38 C the fluid phase did not contain virus.
Only cells could be used to transfer the virus to subcultures. When
the cultures were incubated at 36 C both the cells and fluid phase
contained virus.

To determine if this were the case with the verruca agent, cultures
of AU and monkey kidney cells were incubated at 35 and 37 C. To one
set of three AU and three monkey kidney cell cultures, 0.1 m1 of cell-
free infected wart nutrient fluid was added to each tube. To a second
set of three AU and three monkey kidney cell cultures, 0.1 m1 of a
heavy suspension of infected AU cells in nutrient fluid was added. This

combination (table 12) was again repeated so that one set of three tubes

D7

Table 12: The effect of 35 or 37 C incubation temperature
on the ability of cells or cell-free passage material
to infect AU and monkey kidney cell cultures

 

 

 

 

 

 

 

 

 

Strain Temp. of Cytopathic changes
and Incubation honkey kidney Days
Passage Uo. Inocula (C) AU cells Cells Observed
Cell-free 3S - * - 12
Cells 35 4 as — 7-12
Hood 18
Cell-free 37 - - 12
Cells 37 + — 7-12
Cell-free 3S - - 12
Cells 35 + - 7-12
Oppelt 16
Cell-free 37 - - 12
Cells 37 + - 7-12
Cell-free 3S - — 12
Cells 35 4 — 7-12
Johnson 16
Cell-free 37 - - 12
Cells 37 + 4 7-12

 

* - No cytopathic effect observed.

** + Cytopathic effect observed.

h8

each of AU and monkey kidney cells, infected with cells or cell-free
material, from each of three lines of the agent could be incubated at
35 or 37 C reSpectively. The result of this experiment is given in
table 12. There was no difference, regarding infectivity, whether the
AU cells or monkey kidney cell cultures were incubated at 35 or 37 C.
The cell-free inocula failed to produce cytopathic effect in both cell
lines. Only when infected AU cells were inoculated onto AU cells was
cytOpathic effect observed. It would appear that the verruca agent was
closely associated with the infected cell and unlike the fibroma or
myxoma viruses, the few degrees difference in temperature of incubation
did not release the agent from the cell to produce visible changes in

those cultures receiving fluid phase inocula.

II. Tissue cell culture neutralization tests

 

Although neutralization tests using cells have not given complete
success, some information can be derived from these tests. Tests des-
cribed here were done in an attempt to learn more about the mechanism of
infection from cell to cell rather than in a strict neutralization of the
agent.

In early tests, gamma globulin was used as the neutralizing serum.
Blank and Rake (1955) and our own experience indicated that warts usually
regress at maturity or shortly thereafter. Reasoning that gamma globulin
would contain neutralizing antibodies to warts, dilutions of gamma glo-
bulin were placed in contact with infected cells. The AU cells used were
the Hood strain which had been serially subcultured. The technique used
was the addition of dilutions of serum to heavy suspensions of infected
AU cells suSpended in nutrient fluid. No attempt was made to count the

number of infected cells or determine an infective dose. Serum dilutions

L9

were also made in nutrient fluid i.e., yeast extract medium. The gamma
_globulin used represented a 16 times concentration of pooled adult sera.
The Hood sera were obtained by several bleedings of the same patient from
which the wart tissue had originally been removed. Antisera were also
made by repeated injection of rabbits with AU cells, infected AU cells
and infected cell-free nutrient fluid.

The results of the neutralization tests are presented in table 13.
The contents of the table are averages of several different neutraliza-
tion tests. As controls, normal cells were maintained in nutrient fluid
plus two per cent calf serum; normal AU cells were placed in contact
with a normal sheet of AU cells; infected AU cells were seeded onto
normal AU cell sheets; infected AU cells were mixed with one part normal
calf serum and one part nutrient fluid.

The tests on gamma globulin were arranged so that one volume of
infected cells was mixed with one volume of gamma globulin which had
been diluted to represent 1x, 8x, and undiluted or 16x concentration as
compared to normal serum.

The tests on Hood's (homologous) serum were one volume of infected
AU cells mixed with undiluted, 1:1 and 1:2 dilutions of the serum in
nutrient fluid. All sera were inactivated at 56 C for % hour prior to
use.

All mixtures were incubated 2h hours at h C prior to placing 0.1 ml
of each mixture of cells and serum on several tubes containing monolayers
of normal AU cells. The cultures were observed daily and the cytOpathic
effect, if present, recorded.

Table 13 shows that, compared to the controls, gamma globulin pre-

vented infection of the cells in 16x concentration. bhen gamma globulin

SO

.0mpmasooaa amps» \pooeem oaapmmopao *

 

 

 

 

 

 

 

 

m\H 0\m m\0 m\0 0\m 0\m m\m NH\NH N\m HN\H 0
QH Qm Q0 Q0 0} 0\m Qm NQNH Q0 HQH m
m\0 m\m m\0 m\0 0\0 0\m 0\m NH\0H N\0 Hm\0 :
m\0 0\N m\0 m\0 0\0 0\m m\0 NH\~ N\0 Hm\0 0
Q0 Q0 Q0 Q0 «<0 0\0 Q: 35. Q0 80 m
m\0 0\0 m\0 m\0 0\0 0\0 m\0 NH\H m\0 *Hm\0 H
u
.Haecp .0.0 xma .0.0 x0 .0.0 ea maam0 0a mHHm0 maamo Hopsco0
.Ha0 mua .Ha0 Hua 0cm maam0 04 mHHmo 0a mHHmo 04 co saumm 0a no 0a so Heme 0a
mfiamo 0a Adamo 0a maao0 0a empomeeH empowecH empooecH mamo H«H a Hamo 04 Hamo 0a Hmsuoz
eonamMCH empomecH empowecH maamo 04 empoweeH Hmaaoz em>ammno
UmpommmH mama
Eupwm poem :HHSQoHU magma machpcoo
asymm msomoaosoz mo caaznoaw mssmm 0cm mHHmo De
vmpoowcfl mo mohfipNHE mcfihoamam mpmmp cowpmmfifimppsmc mo mpfidmmm "ma oflfime

51

was diluted to 8x and lx concentration, the infection was delayed for
one or two days. Vith the undiluted Hood serum there also was protection
from infection. Like the gamma globulin, when less concentrated serum
was used, less protection was afforded. The data with both sera empha—
sized the necessity of using large amounts of antibodies to inhibit
infection for when the sera were diluted there was some, but not absolute,
protection against infection. That protection was not afforded by com—
ponents present in normal serum was emphasized by the rapidity with
whicn infected cells and calf serum control cultures showed cytopathic
changes. It would appear from the data presented that gamma globulin,
as well as the serum samples from the patient from whom the wart was
removed, contained antibody against the wart agent.

ihe results of the neutralization tests using rabbit prepared anti-
sera are shown in table 1h. The results of these tests were erratic
and no information could be gained. ihey were included, however, to
present data for discussion.

lhe nature of the neutralization tests employed requires some judg-
ment as to their validity. lhe tests with gamma globulin and the homolo-
gous serum afforded some protection against the infection of normal cells
with infected cells. The test can not, on the other hand, be used as
conclusive evidence of specific antibody in gamma globulin nor the

serum from the patient.

III. Attempts to infect laboratory animals with verruca tissue and wart

 

agent recovered in tissue cell cultures.

 

Because of the danger involved in introducing foreign cells and sera
into humans, animals were inoculated with infected tissues and tissue cell

cultures. Although the AU cells originally came from normal healthy skin

52

.uopmadooza .oz \poommo aflgvmaopho wnwsonm honssz *

 

 

 

 

 

 

 

 

 

Qm Qm Qm Qm Qm Qm Qm Qm Qm Qm e
Qm Qm Qm Qm Qm Qm Qm Qm Qm Qm m
Qm Qm Qm Qm Qm Qm QN Qm Qm Qm s
Qm Qm Qm Qm Qm Qm QH Qm Q0 Qm m
Q0 Qm Q0 Q0 QN QN Q0 Qm Q0 Qm N
m\0 m\H m\0 m\0 m\0 M\0 m\0 m\0 m\0 *MQH a
fill!
Juana muH Hua qua «NH HuH qua mmw: Hua Adonpcoo emswmmno
Edhmm “0.35.5 meHHPSz Show 9360 :5th mHHmO mHHGO D4 whwfl
wwwm-fiamouapc< amenozuwpc< empomenH-Hp:< Hespoz no
maamu empomqu
new mHHmo poaommnH sq

 

 

pagan psownpsq omnmnHHoo umpoomswnflpcm use mHHoo p< Hmsaoc
napnu .mHHoo :4 uopoomnanfipnm panama paw mHHoo :4 Uwpomycw
mo wonspxfia MCHhOHmEo memes :oapunfiamnpSo: Ho mpHSmom «AH capwa

 

53

(Rheeler et al., 1957), the cells had been cultivated in zitrp for
several years. Morphologically they were indistinguishable from the

EeLa cells (Gey et al., 1952) that were originated from neOplastic
tissue. In like manner, the injection of human serum may introduce the
serum jaundice virus. Potentially both the AU cells and the sera used

in the nutrients were capable of producing unwanted results if introduced
into susceptible individuals. In an attempt to circumvent these hazards,
yet appraise the results of the tissue cell cultures, Specimens were

injected into laboratory animals.

A. Monkeys

The animals used were hacaca cvnomolcus and were in C>‘ood health
U k.. b

 

when inoculated. Just prior to inoculation the hair on the abdominal
surface or head was removed. The animals received three types of inocula:
1) Pieces of wart tissue removed from patients. The tissues were placed
into tissue culture fluid containing streptomycin and penicillin over night
at h C before being used as inoculum. 2) Tissue culture cells were used,
i.e. infected AU cells were harvested from cultures after detachment of
the cells from the tube wall. The cells were separated from the nutrient
fluid by centrifugation at low speed and enough supernatant fluid was
permitted to remain on the packed cells so that they could be drawn into
a hypodermic syringe. 3) Cell-free nutrient fluids from infected serial
subcultures. The fluids usually were the supernatant fluids from (2) above.
X-ray "tanning" was done by restraining the monkey and irradiating
a belt 75 x 110 cm across the abdominal area shielding the unexposed area.
The animal received 6 doses of from 100 to 300 r. every second day until

a total of 1000 r. had been applied.

5h

Two animals were injected with cortisone acetate and verruca tissue
or tissue culture material. Each animal received 25 mg cortisone intra-
muscularly five times each week.

Monkey 1 (male, immature). This animal received three specimens
intracutaneously. Fresh verruca tissue smaller than 1 mm3 was drawn into
a syringe in 0.05 ml of basal salt solution. The fragment of tissue was
deposited by forcing the tissue fragment through a large gauge needle
into the skin. At the same time, 0.05 ml of a heavy suspension of infected
AU cells suspended in nutrient fluid and 0.05 ml of infected cell—free
nutrient were injected into the skin at two additional sites.

The observation period on this animal was 7 months. Within seven
to 10 days after injection, the injection site of the tissue culture
fluid and cells was healed and no evidence of wart growth was found. The
site receiving the verruca tissues appeared as a blanched, raised area
for two to three weeks. This was followed by complete absorption of the
foreign tissue. The animal remained healthy during the observation
period and developed no evidence of abnormal tissue growth during the
period.

Monkey 2 (male, immature). Prior to inoculation the animal was
"tanned” by X-ray. The tanned belt was then used as the site of injections
for wart material. All injections were given intracutaneously. The
animal received: 1. Two fragments (less than 1 mm3) of verruca tissue
from two different patients (Fisher.and Bloomquist). 2. (Oppelt strain)
0.05 ml infected AU cell—free fluid, and 0.05 ml infected AU cell sus-
pension (12th serial subculture). 3. (Montgomery strain) 0.05 ml
infected AU cell-free fluid, and 0.05 ml infected AU cell suSpension

(3rd serial subculture). h. (Hood strain) 0.05 ml infected AU cell-free

55

fluid, and 0.05 ml infected AU cell suspension (13th serial subculture).

The animal was observed over seven months. The "tanned" area
soon sealed off, the sites of liquid inocula rapidly healed but the
sites receiving tissue fragments were slower to return to healthy skin.
At no time during the observation period were there any signs or symptoms
of disease observed in this animal.

Monkey 3 (immature). The animal received several small fragments of
fresn verruca tissue (less than 1 mm3) intracerebrally. The needle was
inserted 6 mm below the meninges into the left hemisphere and 0.5 m1 of
saline solution containing the tissue fragments were deposited into the
brain. The tissue used was freshly removed from a patient, age 11
(Hallead), who recently had developed a wart on her finger.

This animal was carefully observed daily for four and one-half
months for evidence of neurotropic involvement. None occurred.

Ronkev h (male, immature). This animal received infected AU tissue
culture materials (dood strain). Pools were made of the 15th through the
26th serial subculture of this strain. Both infected cells and nutrient
fluids were used. Approximately lhO m1 of material was centrifuged in
a horizontal position to separate the cells from the fluid. The cells
were ground in a mortar with a pestle and the liquid phase was then used
to dilute the cell paste. The mixture was again recentrifuged at slow
speed to remove gross particles. The supernatant fluid from slow speed
centrifugation was then ultracentrifuged at L2,0h0 r.p.m. (11h,610x g
for one hour. four tubes containing 35 ml each were ultracentrifuged
and, after the cycle, the top 32 ml of fluid was removed from each tube.
The remaining three ml in each tube was used to rub down the walls of

the tube. There was no visible sediment. The contents from each of the

56

four tubes were pooled and placed into the ultracentrifuge. The second
cycle was one hour at h2,0h0 r.p.m. (110,660x g). The top five ml of
fluid was removed from the remianing tube and the walls of the tube
were rubbed with a glass rod with the final 2 ml of fluid remaining in
the tube. A small insoluble sediment was present in the suspension.
This was removed by slow speed, horizontal centrifugation. The super-
natant fluid was used to inoculate a monkey. Four injections each
consisting of 0.05 ml of concentrated tissue culture material was injected
intracutaneously into four different sites on the abdomen of monkey h.
The animal was observed for seven months. Within ten days the sites of
inoculation healed and no evidence of infection has been observed.

Monkey 5 (male, immature). Two days prior to being used this animal
received 25 mg of cortisone acetate intramuscularly. The cortisone
injections have been continued five times a week and to date (May 10,
1961) the animal has received 2.15 grams total. Fresn verruca tissues,
after being freed from bacteria with antibiotics, were placed intracu-
taneously by trocar. Three circular fragments of wart tissue 1 mm in
diameter and 0.5 mm thick were forced into the skin of the abdomen with
the trocar plunger. The resulting inoculation left small incisions which
were closed by applying a small piece sterile absorbent cotton saturated
with collodion. Introduction of the inocula produced three raised
blanched blebs on the skin. Hithin 3 weeks the blebs had disappeared
leaving only a small scar at the site of inoculations. This animal has
been observed daily when receiving cortisone injections and no growths
have appeared at the injection sites. This animal is still under
observation.

Monkey 6 (male, immature). Two months(59 days) prior to being

injected with verruca material this animal was given 25 mg of cortisone

S7

acetate intramuscularly five times weekly. To date the animal has
received 1.2 grams total and is still receiving the drug. Fifty nine
days after the first injection of cortisone, the monkey received four
site injections of a heavy suspension of infected AU cells in nutrient
fluid. The specimens were original tissue culture passages of wart
tissues from adult patients who have repeated wart infection. Two
intracutaneous injections of 0.125 ml of cells into two sites were made
for each Specimen. Following injection one site for each of the two
Specimens was painted with one per cent (in acetone) 7, 12-dimethylbenz-
alpha-anthracene (Eastman Organic Distillation Products Co., Rochester 3,
New York). This cocarcinogen was applied over the injection Site in

a 10 mm circular skin area. A Single uninjected equal area was also
painted with the drug as a control. This animal healed rapidly at the
site of injection and has been under observations for five days. Obser-

vations will be continued On this animal.

B. Suckling mice

 

Mice have been used almost exclusively in attempts to isolate the
causative agents of neoplasms. The recovery of agents by inoculating
these animals with human tissues has resulted in some skepticism regard-
ing the origin of the isolated agent. An example of these agents is
leukemia by Gross (1951). For this reason, although mice were used, the
emphasis was not placed on the use of this animal Species.

The mice used were virus-susceptible Swiss white. Litters were pooled

with a common mother if necessary to have sufficient number of animals.
All suckling mice were less than three days old when inoculated. The
inoculum consisted of wart tissue recently removed from the patient. The

tissues were either extracted or cut into fragments. A heavy suspension

58

of infected AU cells (Oppelt strain) in the eighth subculture was also
used. In table 15 the number of mice, inoculum, route and observation
period are presented. During the observation period no evidence of
abnormal growth was observed on any of the animals and none died. At

the completion of the experiment all mice were sacrificed and the internal

organs were observed for signs of abnormalities. None was found.

C. Chickens
Fertile white-leghorn eggs were obtained from a commercial source

and were incubated until hatched. One day after hatching, the CAleS
were inoculated with cells from subcultures of three strains of the agent.
Table 16 presents the number of day-old chicks inoculated with the various
infected AU cultures.

0f the 2? chicks inoculated, 1h died during the 60-day observation
period. Those birds ill or dead were examined at autOpsy for evidence
of abnormal growth. Careful examination of the organs of each dead

bird revealed no abnormal growth.

D. Hamsters
The adult hamsters used in these experiments were purchased from

commercial sources. every effort was made to prevent cannibalism of
the inoculated animals but the majority of deaths were due to this cause.
In table 17 the inocula used, the days of observations and the deaths
recorded in adult animals are listed. There were Six deaths in the 20
animals injected with infected AU cells and all were partially or complete—
1y consumed by cage mates. ixamination of internal organs, when possible,
revealed no gross lesions. The observation period on these experimental

animals ranged from 78 to 109 days and no abnormal growths were observed.

Table 15:

59

Results of inoculating mice with fresh verruca tissue
extracts, fragments and tissue cell cultures

 

 

 

Observation

No. wart Volume Period in

Nice Material Patient Route (ml) Days Results
5 Extract Killer I.C.* 0.03 180 Negative
5 Extract Field I.Q.* 0.03 180 Negative
3 Fragments Davis 1.2. 0.03 365 Negative
h Cell Oppelt I.Q. 0.03 365 Negative

culture

 

* I.C. a intracerebral; I.Q. = intracutaneous.

Table 16:

cell subcultures

Day-old chicks inoculated with AU infected

 

 

 

 

No. of Route and Days No. /No.
Chicks Volume (ml) Inoculum Observed Dead/ Inoculated
8 0.01 I.C.* Cells (Hood) 60 h/S
lhth passage
8 0,01 I.C. Cells (Oppelt) 60 5/8
1hth passage
11 0-01 I-C- Cells (Anderson) 60 5/11
original passage
* I.C. = intracerebral.

Table 17:

60

Inoculation of adult hamsters with infected and
non-infected AU cells.

 

 

 

 

 

 

 

 

No. Route and Days Ho. Ho.
hamsters Volume (m1) Inoculum Observed Dead Inoculated
* e ..
S 0.05 I.C. infected ab cells 109 1/5
doos, 17th passage
5 0.05 I.Q.* Infected AU cells 109 h/S
nood, 17th passage
5 (contr 018) None 109 3/5
2 (controls) 0.05 I.C. Normal AU cells 109 0/2
2 (controls) 0.05 1.9. Normal AU cells 109 l/2
5 0.05 I.C. Infected AU cells 78 o/S
dood, 20th passage
5 0.05 ;.o. Infected AU cells 78 1/5
hood, 20th passage
6 (controls) None 78 0/6
h (controls) 0.05 I.C. Normal AU cells 78 O/h
h (controls) 0.05 1.0. Normal AU cells 78 O/h
Summary»
Inoculation No. Deadlgflo. Inoculated Per cent Dead

 

 

Infected EU

Normal AU 0

None

cells

ells

6/20
l/l2

3/ll

 

* I.C. = intracerebral, I.Q. a intracutaneous.

61

The results are summarized at the bottom of table 17. In non—inoculated
control adult animals there were three deaths in the 11 animals. Among
the animals inoculated with normal AU cells there was 1 death. Six of
20 animals died after injections with wart-infected AU cells. Thirty
per cent of the animals receiving infected AU cells died as compared

to 27 per cent of the animals receiving no injections. It would appear
from the results that infected AU cells did not produce growth or death

when introduced into the adult hamsters.

E. Suckling hamsters

 

The suckling hamsters used were laboratory bred from normal animals.
Ten suckling hamsters, one to three days old, were injected intracutaneously
with 0.05 ml of a heavy suSpension of normal or infected AU cells from
the 26th (Hood) passage. The animals were observed f0? 32 to 33 days
without showing any evidence of abnormality. The details of this experi-

ment are illustrated in table 18.

F. Rabbits
Half grown white rabbits were injected intracutaneously with nornal
and wart-infected AU cells. The abdomen was freed of hair with clippers
and each animal received 0.05 ml of heavy suspension of cells. Table
19 presents the data for this experiment.
All animals remained well during the observation period and none

developed lesions at the site of injections.

IV. dlectron microscope observations of tne particles seen in verruca
tissues
Strauss et al. (19L9, 1950) presented electron micrographs represent-

ing the agent causing warts. His crystalline, virus-like cluster of

62

Table 18: Inoculation of suckling hamsters with normal and

infected AU cells

 

 

 

No. Route arri Days LO.
Hamsters Volume in ml Inoculum Observed Dead Inoculated
h 0.05 I.Q.* AU cells, 33 o/h
mood, 26th passage
2 0.05 1.1. AU cells, 32 0/2
dood, 26th passage
2 (controls) 0.05 I.g. Norrel AU cells 33 0/2
2 (controls) 0.05 I.2. Normal AU cells 32 0/2

 

* 1.0. a intracutaneous.

f

Table 19: Results of injecting rabbits with normal AU cells
and infected AU cells

 

 

 

Animal Days
No. Inoculum Observed Results
1 h areas, ultracentrifuge 90 Negative
concentrated cell-free
infected nutrient fluid
(dood strain)
2 Infected AU cells, 90 Negative
hood, 8th passage
3 Normal AU cells 90 Negative

 

63

particles derived from human wart tissues measured 52 mu but when found
independent ranged from 56 - 86 mu. The individual particles were
spherical in shape. Recently, Siegel (1960) attempted to confirm the
work of Strauss et a1. Siegel found uniform particles in extracts from
verruca tissue but found them to measure 16 mu in size.

By following the method described by Strauss et al., verruca tissues
were extracted and limited observations were made on verruca tissue,
infected and normal AU cells.

Electron microsc0pe observations presented spherical particles in
preparations from verruca tissue and infected AU cells as well as a
great deal of debris. The size-range of these particles varied from
50 to 75 mu. The preparations showed particles that were definitely
not uniform, nor were the particles of any one size in abundance.
Preparations of control AU cells did not show particles of the size—

range found in either verruca tissues or infected AU cells.

DISCUSSION

There is no doubt that warts are caused by a filterable agent as
repeated experiments using human volunteers have demonstrated. Although
the injection of volunteers has reproduced wart growth, one common
finding in the experiments was the prolonged incubation period. Usually
it required at least six months after the initial injection before
visual evidence of wart growth was noticed. Another common finding in
the human injection studies was that, after a short growth period, the
experimentally produced wart regressed rapidly.

A comparison of the human injection studies with the results ob-
tained in tissue cultures inoculated with wart material cannot be made.
The hosts' cells are not all destroyed by any infectious agent because
of the hosts' many defensive mechanisms. In in yitgg cultures, cells do
not have the benefit of this protection and infection is rapid and over-
whelming. Virus activity nay not always be visible as cytopathic effect.
Virus may enter the cell producing a latent infection or at any time
may become cytocidal. Why the agent was cytocidal, i.e. producing cyto-
pathic effect, when wart tissue cells were placed in contact with AU
cell cultures and not with other epithelial cell lines is not known.

The AU cell line originally came from normal skin, but so did the Hus
3075 line of epithelial cells. The former required a relatively simple
growth medium while the latter required a complex growth medium, and

it is possible that the nutrient requirements of the two different cell
lines resulted in difference in susceptibility.

The reaction observed when wart tissue cells were placed in contact

with AU cell cultures was first thought to be due to cytotoxic substances

6h

65

in wart tissues. Twenty to thirty subcultures of three strains (Hood,
Johnson and Oppelt) eliminated the possibility that this was the cause
of cell degeneration. Further evidence that the agent isolated was
associated with wart tissues was obtained by isolating the agent from
22 (30.h3 per cent) of 69 different individuals.

The agent isolated from wart is the first such agent derived from
growths of man. It appears from the work reported here that cell to
cell transfer of the agent occurs since the experiments designed were
not able to show infection with cell-free material. With agents producing
tumor growth in animals, cell-free fluids have been shown to be infectious.
The pattern shown by the wart agent and that of lymphomatosis in chickens
is not dissimilar. Injections of tumor cells into susceptible birds rapidly
results in a tumor growth at the site of injection with metastases and
ultimate death of the bird. When on the other hand, cell-free extracts
of the tumor are injected, an incubation period of 100 to 300 days is
required before evidence of disease appears. The tumors developing in
the latter case are diffused and generalized instead of a tumor mass.

Many diseases suspected of being of viral etiology have presented
difficulties in isolating the agent. This difficulty has been overcome
in some cases by placing diseased cells from the patient directly in
contact with tissue cell cultures. Rowe et a1. (1958) in this manner was
able to isolate the salivary gland virus from children.. Subcultures
of the agent required that infected cells be transferred as no evidence of
infection 12.XEE£2 was observed when cell-free inocula were used. The
agent of cytomegalic inclusions isolated by‘fieller et al. (1957) also
required cell transfer initially but eventually cell-free fluids were

shown to be infectious to tissue cell cultures. Weller, Witton and

66

Bell (1958) isolated the viruses of varicella and herpes zoster in
tissue cell culture and again infected cultured cells were required to
subculture the agent.

Stewart and Irwin (1960) described several agents isolated from
neoplastic tissues in monolayer cell cultures. Although the original
starting material was cell-free, the agents isolated from 3 different
individual neoplastic tissues required cells to transfer the agent. All
of these references support the theory that virus exists in cells and
can be transferred to daughter cells without total destruction of the
cells in the cultures. This was best illustrated by Temin and Rubin
(1959) with Rous sarcoma virus.

The isolation of disease—producing agents from several different
types of diseases, hitherto unsuccessful, has been accomplished by using
the infected cell as the source of the agent. It can be predicted that
many more agents, especially those associated with tumors, will be
isolated by utilizing the cell to cell transfer of the agent within the
tissue cell culture system.

The attempt to determine the type of immune response in wart
infection was inconclusive. Gamma globulin and homologous serum contained
an antibody which prevented infected cells from infecting normal cultured
cells. The exact mechanisms of this inhibition of infection probably
can be explained by the results obtained by Russell (1961) in which
complement fixing antibodies were found against the wart agent in the
patients' sera. Under these circumstances, the wart agent and antibodies
appeared in the same individual. Spontaneous regression of warts usually,
but not always, takes place at puberty (Blank and Rake, l9Sh) but it has

not been determined whether this is due to antibody formation or other

67

causes such as local tissue resistance.

Animals injected with wart cells, infected tissue culture cells and
cell-free nutrient fluids failed to produce any evidence of infection.

If the agent is intimately associated with the cell as was shown by the
tissue cell cultures it is not surprising that infection was not
established in the animals. With this close relation between cell and
agent, the transfer of the cells to a foreign Species would result in
immediate rejection of the tranSplant of cells. Cell-free inoculum
containing the agent would also find adsorption and certainly penetra-
tion of the agent into foreign cells impossible.

The electron microscope studies reported do nothing to settle the
differences as reported by Strauss et a1. (l9h9, 1950) and Siegel (1960).
It is sufficient to state that particles were observed in extracts from
wart tissue cells and infected AU cells but not in normal AU cell extracts.
The significance of these particles to the infective agent can only be

speculated.
ADDENDUM

In April, Nendelson and Kligman (1961) reported that they had
successfully isolated the virus of warts in monkey kidney tissue cells.
Inoculation of human volunteers with cell-free nutrient fluids from the
cultures produced wart growth in the skin of the volunteers. This is
the first successful report of isolating the wart agent using monkey
kidney cell cultures although others (Siegel and Novy, 1955) have attempted

isolations as described by Mendelson and Kligman without success.

SUI'J‘ARY

1. More than 200 samples of verrucae from 195 patients were tested
in 13 different types of tissue cell cultures in an attempt to isolate
the wart agent.

2. Only when small fragments of fresh verruca tissue were placed
in contact with AU cell cultures was cytopathic effect observed.

3. By cell to cell transfer three strains of the verruca agent
were carried through 23, 26, and 32 serial subcultures respectively.
In addition, the agent was successfully isolated from 30 per cent of
69 verruca samples.

h. Attempts to establish new human skin and verruca cell culture
were unsuccessful.

5. Human gamma globulin and homologous patient's sera, from a
single patient, were found to inhibit cell to cell infection.in zitgg
in a cell—serum neutralization test. Diluted patient's serum or gamre
globulin failed to give protection to normal cells indicating that
large amounts of antibody were necessary to prevent infection.

6. Monkeys, rabbits, hamsters, mice, and chickens were injected
with verruca cells, infected AU cells, and cell-free material. Hone

gave evidence of infection.

68

BIBLIOGRAPHY

ANDERSON, W. A. D. 1957 Pathology, 3rd ed. pp. hl7-1158. The U. V.
Mosby Company, St. Louis.

ELARD, D. ‘.-7., G. S. BdAUDLh‘AU, R. A., BONAR, D. G. SAARP, AID J. T. BulARD
1957 Virus of avian erythroblastosis. III. Antigenic constitution
and relation to the agent of avian myeloblastosis. J. Natl. Cancer
Inst., 18, 231-259.

-ITTNnR, J. J. 1936 Some possible effects of nursing on the mammary
cancer gland tumor incidence in mice. Science, 8b, 162.

BIVINS, J. A. 1953 The growth in the developing chicken embryo of a
filterable agent from verruca vulgaris. J. Invest. Dermatol., 29,

h7l-h81.

BLANK, a., AN? c. RAKE 1955 Viral and rickettsial diseases of the skin,
eye, and mucous membranes of man, pp. 156-181. Little, brown, and
Company, Boston.

 

 

BORREL, A. 1903 Epithelioses infectieuses et epitheliomas. Ann. inst.
Pasteur, 17, 81-122.

EURMESTER, B. R. 1955 In vitro and in vivo neutralization of the virus
. . —. -a W'- -—"._ a ,
of Visceral lymphomatOSls. rroc. Doc. nxptl. 8101. Led., 90, 28h-286.

 

csssvsa, F. 8., AND G. A. JANEWAY 19h1 Immunity induced against the
Brown-Pearce carcinoma. Cancer Research, 1, 23-27.

CIUFFO, G. 1907 Innesto positivo con filtrato di Verruca Volgare. Gior.
ital. d. mal. vener., h8, 12-17.

CLAUDi, A., AND J. B. NURPHY 1933 Transmissible tumors of the fowl.
Physiol. Rev., 13, 2&6-275.

DMOCHOYSKI, L. 1959 Viruses and tumors. Bacteriol. Revs., £3, 18—b0.
DMOCHOUSKI, L. 1961 Viruses and tumors. Science, 133, 551-561.

DULPnCCO, R., AN“ M. VOGT l95h Plaque formation and isolation of pure
lines with poliomyelitis viruses. J. Exptl. Med., 22, 167—182.

EAGLn, n. 1955 Nutrition nerds of mammalian cells in tissue culture.
Science, 122, 501-50h.

BCKERT, E. A., D. C. SHARP, D. V. DEART, I. semen, AND J. V. BLARD 1955
Virus of avian erythromyeloblastic leukosis. IX. Antigenic consti-
tution and immunologic characterization. J. Natl. Cancer Inst., 16,

593-6h3.

69

7O

EDDY, B. 5., S. L. STEMART, R. IOUN7, AND 3. B. NIDLR 1958a NeOplasms
in hamsters induced by mouse tumor agents passed in tissue culture.
J. Natl. Dancer Inst., 20, 7h7-76l.

odor, B. s., S. 8. STLVART, AND U. EnKnLNI 1958b Cytopatnogenicity in
tissue cultures by a tumor virus from mice. Proc. Soc. hxptl. Biol.

d., 9__8_-, 8118- 851.

ADDY, B. 1., If. P. 5101.15, J. If. nl'lR‘iLEl’, S. 2.. S‘lnlli‘ni'l', AID R. J. ALL“. EILJR
1958c hemagglutination with the SE polyoma virus. Virology, 6, 290-
291.

induction of tumors in rats by tissue culture preparations of 3C poly oma
virus. J. Natl. Cancer Inst., 22, 161-171.

AL '“”“, V., AND 0. HANG 1908 Axperimentelle Leukaemie bei huehnern.
Zentr. Bakteriol. Parasitenk., Abt. I. Orig., A6, 595-609.

Growth in tissue culture of cytOpathogenic agent from strain of virus
which produces avian lymphomatosis. Proc. Soc. dxptl. Biol. Ned.,

21. ass—€57.

£31433, 0. 1957a Cell- free trans mission in adult Swiss mice of a cisease
having the character of a leukemia. J. nxptl. Ned., 105, 307-318.

.lIshD, C. 1957b Leukemia of adult mice caused by a transmissible agent.
Ann. N. Y. Acad. 3ci., 68, 522- 533.

EURTd, J., R. E. EUEFDTT, N. BANSSINUICZ—RCDRIGUDZ, AND A. C. UPTCN 1956
Character ofa agent inducing leukemia in ne~born mic:3 Proc. Doc.
Exptlo BiOlo LIEd o, 93, 165-172.

SURTA, J., AND N. L. STUEBS 193h Tissue culture studies on relation of
sarcoma to leukosis of chickens. Proc. Soc. nxptl. Biol. hed., £9,

379-h26.

GEE, G. 0., b. 33. COEECAX', AND E. T. KUBICDEC 1952 Tissue culture studies
of the proliferative capacity of cervical carcinoma and normal epithe-
lium. vancer Research, 12, 26h-265.

GCRM , P. A., AND L. V. LAN 19h9 an attempt to dernonstrate neutralizing
antibodies to the mammary tumor' 'milk agent" in mice. Erit. J. Cancer,

,3. 90-93.

35035, L. 1951 "Spontaneous" leukemia developing in C33 mice following
inoculation in infancy with AK-leukemic extracts, or AK embryos. Proc.
Soc. dxptl. Biol. Ned., 76, 26-32.

sauna, J. h., AND R. s. UALLACE l9h9 Relation of oxygen and temperature
in the preservation of tissues by refrigeration. Proc. 30c. exptl.
3101. Dlfid o, 71, 196-200.

71

JADASSOHN, J. 1896 Sind die Verrucae vulgares ubertragbar? Verhandl.
dermat. gesellsch., 5, N97.

KABAT, E. A., AND J. FURTH 19hl Neutralization of the agent causing
leukosis and sarcoma of fowls by rabbit antisera. J. Exptl. Ned.,

11., 257-261.

KILHAM, L. 1959 Virus transformation and cancer. In Perspectives ip
Virolo , pp. Sh-7h. edited by N. Pollard. John Wiley a Sons, Inc.,
New York.

 

KINGERY, L. B. 1921 The etiology of common wart. Their production in the
second generation. J. Am. Med. Assoc., 16, th-hNZ.

KARKIN, M. F. 1959 The establishment of a strain of human cells in
tissue culture. Austr. J. Cxptl. Biol. Med. Sci., 37, 505-508.

LAN, L. N., AND R. A. NALNGRNN 1951 Studies on the "cytotoxic" property
of antiserum of the mammary tumor agent. J. Natl. Cancer Inst., 11,

1259-1268.

Lo, N. H. r., a. 0. car, AND P. SnAPRAS 1955 The cytopathogenic effect
of the Rous sarcoma virus on chicken fibroblasts in tissue cultures.
Bull. John nopkins dosp., 21, 2h8-256.

PULLIVJGRLSI‘I, R. A. , G. h). BENEIISON, B. R. AI‘IDSRSON, AND C. C. RISLEY 1951
Serologic study of the microsome fraction of normal and neoplastic
mouse tissues. J. Natl. Cancer Inst., 11, 1277-1286.

MCQUILK J, N} T., V. J. EVANS, AND N. R. EARLS 1957 The adaptation of
additional lines of NCTC clone 929 (Strain L) cells to chemically
defined protein-free medium NCTC 109. J. Natl. Cancer Inst., 19,

885-907.

NdNDELSON, C. G., AND A. M. KLIGLAN 1961 Isolation of wart virus in
tissue culture. Arch. Dermatol., 82, 559-562.

MIRAND, E. A., D. T. MOUNT, G. E. MOORE, J. T. GRACE Jr., AND J. E. SOKAL
1958 Induction of tumors by a virus-like agent(s) released by tissue
culture. Proc. Soc. Exptl. Biol. Ned., 22, 1-5.

DE MONEREUN, N. A., AND F. I. GCCDPASTURD 1932 Infectious oral papillo—
matosis of dogs. Am. J. Pathol., 8, 83-56.

MORGAN, J. 3., N. J. NORTON, AND R. C. PARKNR 1950 Nutrition of animal
cells in tissue culture. I. Initial studies on a synthetic medium.
Proc. Soc. nxptl. Biol. Ned.,‘12, 1-8.

OLSON, 0., A. n. PANUKCU, D. r. BROES“, T. KONALCZYK, a. J. SATTER,
AND J. N. NICE 1959 A urinary bladder tumor induced by bovine
cutaneous papilloma agent. Cancer Research, 12, 779-782.

72

PIERRE, P. V., V. J. EVAI-IS, W. R. EARLS, Cr. 1!4'... ri'XAfL'T, AND W. C. BEDEIL
1956 Long-term tissue culture of human skin. Am. J. Hyg., 63,
52-58.

ROBERTSON, H. 5., K. T. BRENNER, AND J. T. SYVNRICN 1955 Propagation
$3 vitro of poliomyelitis viruses. VII. pH change of HeLa cell
culture for assay. Proc. Soc. EXptl. Biol. Med., 66, 119-122.

ROUS, P. 1911 lransmission of a malignant new growth by means of a
cell-free filtrate. J. Am. Med. Assoc., 56, 198.

RODS, P., J. B. MURPAY, AND N. H. TITLER 1912 A filterable agent in
the cause of-a second chicken-tumor, an osteocnondrosarcoma. J. Am.
lied. Assoc., _5__9_, 1793-17921.

ROUS, P., AND J. H. BLARD 1935 ihe progression to carcinoma of virus-
induced rabbit papillomas (Shops). J. Exptl. Med., 63, 523-5h8.

BONE, h. P., J. h. HARILEY, H. 3. CRANBLEHT, AID F. M. MA31ROLA 1957
Detection of hunan.salivary gland virus in the mouth and urine of
children. Am. J. hyg., 61; 57-65.

ROEJE, w. P., J. 1:. 111111.31, I. BRCDSKY, AND R. J. swarm 1958 Comple-
ment fixation with a mouse tumor virus (SE polyoma). Science, 128,

1339—13h0.

RUSSELL, J. N. 1961 Complement fixation studies with verruca vulgaris.

m. S. Thesis, Michigan State Univ.

SLRRA, A. 1908 Ricerche istologiche e sperimentali sul condiloma
acuminato - I papillomi del capo e la verruca volgare. Gior. ital.
d. mal. vener., 62, ll.

SnARPLfiSS, G. R., V. DNERNDI, AND a. R. COX 1958 Cultivation in tissue
culture of the virus of avian lymphomatosis. Proc. Soc. Exptl. Biol.

MEX} - 2 22: YES-'75? .

SAOPE, R. E. 1932a A transmissible tumor—like condition in rabbits.
Jo mptlo Ivjedo, gé, 793-8020

SHOPS, R. d. 1932b A filterable virus causing a tumor-like condition
in rabbits and its relationsnip to virus myxomatosum. J. EXptl.
Had”, E, 803-8220

SIOPN, R. E., R. NANCOLD, L. C. NacNANARA, AND K. R. DULBRLL 1958 An
infectious cutaneous fibroma of the Virginia white-tailed deer
(Odocoileus virginianus). J. mXptl. Hed., 108, 797-802.

smasL, B. V., Am F. s. NOVY, Jr. 1955 Cultivation studies on wart
suspensions of verruca vulgaris and condyloma acuminatum. J. Invest.
Dermatol., 25, 265—268.

SIEGLL, B. V. 1956 Contaminant filterable agent derived from a human
wart. J. Invest. Dermatol., 21, 379-381.

73

SIBGNL, B. V. 1960 Blectron microscopic studies of the virus of human
warts. J. Invest. Dermatol., 25, 91-93.

STEWARI, S. E. 1953 Leukemia in mice produced by a filterable agent
present in ARR leukemia tissues with notes on a sarcoma produced by
the same agent. Anat. Record, 117, 532.

SldNARl, S. 3., B. B. EDDY, A. N. GOCRRNOUR, N. G. BORGESS, AND G. E.
GRUBBLS 1957 Induction of neoplasms with a substance released from
mouse tumors by tissue culture. Virology, 2, 380-h00.

SIJNARI, S. 5., B. B. EDDY, AND N. G. BORQNSE 1958a Neoplasms in mice
inoculated with a tumor agent carried in tissue culture. J. Natl.
Cancer Inst., 29, 1223-1236.

STEWART, S. N., B. B. EDDY,.AND N. F. SIANTON 1959 Induction of neo-
plasms in mice and other mammals by a tumor agent carried in tissue
culture. Proc. 3rd Canad. Cancer Conf., pp. 28L-305.

S‘ifl'fiAR‘l, s. s., AID B. E. 2931’ 1959 Properties of a tumor—inducing
virus recovered from mouse neoplasms. In Perspectives Ea Virology,
pp. 2h5-255. edited by H. Pollard. John Liley & Sons, Inc.,

New York.

 

SISEART, S. 3., B. E. NDDY, AND N. E. SIANICN 1960 Progress in virus
research - The polyoma virus. In Provress 1E Experimental Tumor
Research. pp. 67-85. Edited by F. Romsurger. J. B. Lippincott
Company, Philadelphia.

 

SIJTARl, S. 5., AND N. L. IRWIN 1960 Cellular proliferation in primary
tissue cultures induced with a substance derived from cell-free
concentrates from human neoplastic material. Cancer Research, 20,

766-767.

STRAUSS, N. J., H. DENYING, AND J. L. NSLNICK l9h9 "Crystallin" virus-
like particles from skin papillomas characterized by intranuclear
inclusion bodies. Proc. Soc. EXptl. Biol. Ned., 72, h6-50.

SlRAUDS, M. J., R. ELNIING, AND J. L. NELNICK 1950 Virus-like particles
and inclusion bodies in skin papillomas. J. Invest. Dermatol.,

15, h33-hhh.

TENIN, d. N., AND H. RUBIN 1959 A kinetic study of infection of chick
embryo cells 12 vitro by Rous sarcoma virus. Virology, 8, 209—222.

TNEILER, N. 1937 Spontaneous encephalomyelitis of mice, a new virus
disease. J. Exptl. Ned., 65, 705-719.

ULBRICH, A. P., N. U. ARNNDS, AND D. KOPRINCE 1957 {Vartsz Jiscussion,
with special reference to therapy by total enucleation. J. Am. Osteo-
pathic Assoc., 56, 3L5-3h9.

7h

1'

:GLLZR, I. P., h. A. VIEIC , AR) J. PeLL- 1958 The etiolneic scent of
varicella and herpes zoster. Isolation, propagation, and cultural
characteristics in vitro. J. mxptl. Ned., 108, 8h3-868.

NALLBR, T. 5., AND R. M. EITlCN 1958 The etiologic agents of varicella
and herpes zoster. Serologic studies with the virus as propagated 12
vitro. J. Exptl. Ned., 108, 869-890.

URBLLER, C. E., C. N. CANBY, AND E. P. CAbLjY 1957 Long-term tissue
culture of epithelial—like cells from human skin. J. Invest. Dermatol.,

22, 383-392.

NILE, U. J., AND L. B. KINGRRY 1919 lhe etiology of common warts.
Preliminary report of an experimental study. J. Am. Med. Assoc.,

12, 970-973.

NOOLLEY, C. V., AND N. C. SHALL 1956 Experiments on cell-free transmission
of mouse leukemia. Cancer, 2, 1102-1106.

HICHI Q

N!

 

N STRTE UNIV. LIBRRRIES
VUIIIHIIllWllllllllINII!MllHlllHlllllllllHl
3 29301 1062530