STUDIES ON THE RELATIONSHIP OF THE HEPATITIS VIRUS
TO PERSISTENT SYMPTOMS, DISABILITY, AND HEPATIC
DISTURBANCE ("CHRONIC HEPATITIS SYNDROME")
FOLLOWING ACUTE INFECTIOUS HEPATITIS 1
By JOHN R. NEEFE,2 JOSEPH STOKES, JR., ROBERT S. GARBER,

AND

SYDNEY S. GELLIS
(From the Nutritional Service of the Department of Pediatrics (Medical School) and of the
Gastrointestinal Section (Medical Clinic of the Hospital), University of Pennsylvania)

The term "chronic hepatitis" has been suggested
by Barker, Capps, and Allen (1) for reference to
those patients with infectious hepatitis who fail
to recover within 4 months of the onset of the
disease. This term also appears to be applicable
to those, patients with homologous serum hepatitis
who fail to recover within 4 months. As used
here the word "chronic" refers only to "duration
without implication concerning the nature of the
pathologic process or the eventual prognosis."
The frequency with which "virus hepatitis"
becomes chronic is not known and probably varies
with the patient's age, general condition (nutritional and otherwise), management, and possibly with the strain of virus concerned. However,
in a series of 431 unselected cases diagnosed as
"infectious hepatitis" in the Mediterranean
Theater (1), 18 per cent were classified as chronic
on the basis of the above definition. Furthermore,
under the favorable conditions associated with
the study of induced infectious hepatitis in one
group of healthy, well-nourished, adequately
treated volunteers between the ages of 18 and 32,
approximately 15 per cent failed to recover completely within 4 months, although all achieved
complete clinical recovery within one year (2, 3).
On the basis of these data and the high incidence
of acute virus hepatitis during recent years, the
occurrence of a relatively large number of such
chronic cases (as defined above) might be expected.

Chronic hepatitis presents a number of important medical problems. Recognition frequently
may be difficult, particularly in the non-icteric
cases, and a lack of knowledge of the factors leading to chronicity has prevented a fundamental
approach to its prevention and treatment. Furthermore, the chronic form of the disease may be of
importance in the epidemiology of virus hepatitis,
particularly when unrecognized.
The relationship of the hepatitis virus to the
chronic stage of the disease unfortunately is obscure. A knowledge of this relationship obviously
is essential for the clarification of some of the
problems mentioned. For this reason, it was considered desirable to attempt to isolate the hepatitis virus from patients with a chronic form of
the disease. Suitable materials for study were
available from three volunteers with chronic active
non-icteric hepatitis observed among a group of
volunteers in whom acute infectious hepatitis had
been induced under experimental conditions by
the oral administration of feces known to contain
the virus of infectious hepatitis (virus IH) described in previous reports (4, 5). It is the
purpose of this preliminary report to describe the
results of attempts to demonstrate the hepatitis
virus in serum, feces, and liver tissue obtained
from these three chronic cases.
MATERIALS AND METHODS

investigation was made possible by a grant
from the Donner Foundation, Inc., Philadelphia, Pa., and
also was aided in part by the Commission on Measles and
Mumps, Army Epidemiological Board, Preventive Medicine Service, Office of the Surgeon General, U. S. Army,
Washington, D. C.
2 National Research Council Senior Fellow in the Medical Sciences.
3 The term "virus hepatitis" is used to include both infectious hepatitis and homologous serum hepatitis.
1 This

Source and materials. The histories of the three volunteers with chronic active non-icteric hepatitis have been
described elsewhere (3) and do not warrant repetition
here. The diagnosis appeared to be unequivocal and was
based on the history, symptoms, laboratory evidence of
hepatic disturbance, and, in two of the cases, on the histologic findings in liver biopsy specimens obtained 6 and 9
months respectively after the onset of acute hepatitis.
The same strain of virus had been used for the induction of
acute infectious hepatitis in all three cases. Specimens
of feces and serum were collected at intervals through-

329

330

JOHN R. NEEFE, JOSEPH STOKES, JR., ROBERT S. GARBER, AND SYDNEY S. GELLIS

biopsy specimen obtained surgically from Case 2 (HPZ)
at the time indicated in Figure 2 (approximately 6 months
after the onset of hepatitis). Serum and feces specimens
were frozen within 1 hour after collection. They were
stored at - 10° to - 200 C. until the time of administration to volunteers, with the exception of a few hours

out the course of the disease. The times of collection of
the individual serum and feces specimens included in the
pools used in the present study are shown in Figures 1,
2, and 3 in which the results of serial hepatic tests roughly
reflect the course of the disease in the three cases. The
liver tissue tested for the presence of virus was part of a

CHRONIC HEPATITIS-D.C.H.
DAYS POST INOCULATION

S. BILIRUBIN

10 0

40

601:
6.0

E

4.0
2 0°

CEPHALIN

46

S

I

260

240.
I

-

I

520

.320

400

440'

I

I

.
5. .I

r~~MO

.b

r

r

A~~~~~~~~~~~~~~~~~

III
JI

4+

3,
2 *.
I

HOUt READING

+

-

10.0
9.0

30 MINUTE

SO
70O
6.0

__a_

IA,
N

0

THYMOL TEST
TURSOID1TY

I

__OIW

.1.0

FLOCCULATION

I

S SS

W~~~

_

0

20

660

620

.111
~~~

I

3

MOMS. PCR
100 MLS.

60

I
I
I
I
I
I

901

IL

Is

.W4~~~~~~~\1A

S0O
4O

.3.0
20
I

.ALAAAA
%-"v
Am!

'%

O

0

THYMOL TEST
I0

HOUR f LOCCULATION

COLLOIDAL GOLD
24 HOUR READING

3i
I

1

APT

RAFV
_'

0

0

BROMSULPHALEIN 2°
20

*PER CENT RETENTION
-AT 45 MINUTES

1S
10
5
0

S. GLOBULIN
GRAMS PER
100 MLS

35
30
25
20

r n Lt 2 - S

~vM--

I5

SEDIMENTATION
RATE

30

MM. PER'HOUR

10

20
I'5

0
I

DAYS POST INOCUJLATION

-20

0

40

s0

120

160

200

240

200

320

360

400

0640

FIG. 1. RESULTS OF SuAIAL HEPATIC TESTS IN VOLUNTEER D. C. H.
The "S" and "F" located beneath the time scale at the top of the figure indicate the times when serum (S) and feces (F) specimens were collected. Acute hepatitis had been induced in this subject by
the oral administration of a feces preparation known to contain a representative strain (virus IH, Pa.) of
infectious hepatitis virus. For approximately one year after the disappearance of jaundice associated
with the acute hepatitis, he had persistent symptoms resulting in partial incapacitation. A microscopic
examination of the liver biopsy, obtained at the indicated time, revealed the stroma of many of the portal
triads, to be infiltrated with excessive lymphocytes, plasma cells, and histiocytes.

331

STUDIES ON THE HEPATITIS VIRUS

CH'RONIC HEPATI SS-H. P. Z.
DAYS POST INOCULATION

S. BILIRUBIN
PER
MGMS.
100 MIL5

o:

0

40

i

ONSET

60

120

I60

245 0

200

260

320

5110S5

5.0

F

3.0
2.0_

..0

0

CEPHALIN
FLOCCULATION

3

IF-____A

+

46 HOUR READING

O*

THYMOL
TURBIDITY

10.0 _

9.01
7.0-

6.0-

30
3.0

-

--I

20-

I0

THYMOL
FLOCCULATION

4

III

/W

_

All

-

---.

COLLOIDAL GOLD
0

BROMSULPHALEIN

.S
30F

PER CENT RETENTION
AT 45 MINUTES

S. GLOBULIN
GRAMS PER
100

MLS

SEDIMENTATION
RATE
MM. PER HOUR

DAYS POST INOCULATION

20

20

3r

1

NI
3.02.5-1
2.0 -

30
2

-

4

0

201

0

s0

40

520

160

200

20

80

320

360

FIG. 2. RESULTS OF SERIAL HEPATIC TkSTS IN VOLUNTEER H. P. Z.
Acute hepatitis was induced as described in the legend of Figure 1. For other data referable to the figure, see legend of Figure 1. A part of the liver.biopsy obtained at the time
indicated was used in the present studies (see text). This volunteer continued to have symptoms and partial disability for approximately 10 months after the onset of the acute attack.
day when they were thawed simultaneously, the
pools prepared, and then refrozen. The liver tissue was
placed in a dry bottle and was frozen within 15 minutes
after its surgical removal. It also was stored at 100
to -20° C. until used.
Preparation of materials: Feces pool 13-14-15 FIH.
The feces specimens from the individual cases were
thawed and a portion of each was pooled with the others
from the same case, yielding pools 13, 14, and 15 respectively. Sufficient distilled water was added to each
of these pools to produce, after mixing, the consistency of
on one

-

thick paste. Additional distilled water then was added
and a heavy homogeneous suspension was obtained by
thorough mixing in a Waring Blendor. This then was
strained through gauze to remove any remaining large
particles. Each of the 3 pools (13, 14, 15) then was cultured (no commonly pathogenic intestinal bacteria were
isolated), and, after the culture reports were available,
equal portions from each were pooled and mixed to yield
feces pool 13-14-15 FIH. The final pool thus included
a total of 23 feces specimens obtained 92 to 342 days
after the initial onset of hepatitis (Figures 1, 2, and S.).

a

332

JOHN R. NEEFE, JOSEPH STOKES, JR., ROBERT S. GARBER, AND SYDNEY S. GELLIS

CHRONIC 'HEPATITIS-R. H. M.

OAYS POST INOCYAti0N
65.0

S. BILItUBIN

50.0

0.o
S..
7.0

moms$ PEt
0oo Mo.&

0.0

S.o

4.0
3.0
a..

*.0
0

CEP,HALIN
FLOCCULATION4
4$ HOUR READING

THYMOL
TURBIDITY

4.

-it'i

3.

2*
3,

0

50.0

9.0
6.0
7.0

i--2I

.6

'SO
4.0
3.0
2.0
1.0
0

THYMOL
F LOCCU LATION

, + -~I
___ --t

2*

0

COLLOIDAL GOLD

B ROM SULPHALEIN
PERt CENT RETENTION
AT 45 MINUTES

S. GLOBULIN
GRAMS PtR
100 ML3.

e

-

4*

20

s
0.

4.0
3.5
. 3.0
2.S
2.0
It 9

SEDIMENTATION
RATE
MPA PER HOUR

Sp
,40
30

20
I0
0

OAYS POST

INOCULATIOI.

-20

0

40

so

520

200

.100

240

2o0

320

360

400

440;

FIG. 3. RESULTS OF SERIAL HEPATIC TESTS IN VOLUNTEER R. H. M.
See legend Figure 1 for the other data referable to this figure. This subject continued to have persistent symptoms and partial disability for approximately 10 months after the onset of acute hepatitis.
Serum pool 1-CSIH. Approximately 2 ml. of each
serum specimen obtained from 106 to 367 days after the
onset of hepatitis were mixed together to form this pool
which included a total of 15 serum specimens (Figures

1, 2, and 3).
Liver suspension 1-CLIH. The biopsy specimen of
the liver tissue, estimated to weight between 100 and 200
mgm., was thawed and promptly was ground with sterile
alundum. The ground liver tissue then was suspended
in approximately 12 ml. of sterile beef heart infusion
broth. The suspension was centrifuged for 10 minutes at

2,000

The supernatant then was removed and
100 C.
Just prior to its oral administration to the volunteers,
the supernatant was thawed and diluted to approximately
100 ml. with sterile physiological saline solution and this
(liver suspension 1-CLIH) was divided into equal portions (20 ml.) which were ingested by each of the 5 subr.p.m.

stored at

-

jects.
The report of Colonel Balduin Lucke, of the Army Institute of Pathology, Washington, D. C., on the microscopic appearance of this liver tissue was as follows:

333

STUDIES ON THE HEPATITIS VIRUS

"The hepatic structure is preserved but allowance must
be made for variations in lobular configuration that are
normal at the surface of the liver. The stroma of most
portal triads contains a moderate excess of cells, mostly
lymphocytes and histiocytes; some triads, however, are
entirely normal. No connectiye tissue proliferation is
evident. Occasionally minute foci of cell reaction similar
to those of the triads are met within the interior of some
lobules. Many liver cells are swollen and appear to be
laden with glycogen; these cells are often binucleated.
It is probable that they represent regenerated cells.
About half of the liver cells seem to have a low glycogen
content, suggesting that storage of this material has
varied in different parts of the tissue."
Volunteers. All of the men who volunteered as subjects for the study were male inmates of the New Jersey
State Prison, Trenton, N. J. Because of statistically
significant evidence (2) indicating that susceptibility to
the virus of infectious hepatitis used in this study decreased after the age of 26, and decreased rapidly after
the age of 30, an effort was made to obtain volunteers
under 30 years of age. Unfortunately, only a few of the
volunteers within this age range could be made available
at the time. It was necessary, therefore, to use a number of men who were not within the age range of greatest
susceptibility (up to 26 years of age) to this strain of
virus. The ages of the 5 men who were inoculated with
the feces pool were 23, 27, 31, 32, and 33 respectively.
Those inoculated with the serum pool were 20, 29, 31, 32,
and 39 respectively. Those who received the liver suspension were 21, 28, 29, 34, and 34 respectively. All were
considered to be in good health. None had a history of
previous jaundice or presented clinical evidence of
hepatic disease prior to inoculation. All of the men were
isolated as a group in a special ward provided by the
New Jersey State Hospital, Trenton, N. J. The period
of isolation began 1 month prior to inoculation and continued for 2 months after inoculation. During this period, the only contacts were with the nurses, the special
guards assigned by the prison, the technicians, and the
supervising physicians. None of these contacts reported
any illness during the period of experiment. Although
personal contact between the volunteers was not preventable at meal and recreation times, they were quartered
in individual rooms with individual toilet facilities and
individual eating utensils were used. Registered nurses
were in constant attendance during the isolation period.
Following their release from the isolation ward at the
New Jersey State Hospital, the men were returned to the
New Jersey State Prison where they were followed for an
additional 3-month period.
Studies on volunteers. The hepatic studies described in
previous reports (3, 6, 7) were conducted on each subject at frequent intervals both before and after inoculation.
Total and prompt direct-reacting (1') serum bilirubin
determinations, urine bilirubin and urobilinogen tests, and
the cephalin-cholesterol flocculation, colloidal gold, and
thymol tests were conducted at least twice weekly during
the period of observation. Bromsulphalein tests were
done once weekly. Serum total protein, albumin, and

globulin were determined at less frequent intervals.
Oral temperatures were recorded at least twice daily.
With the appearance of any symptoms or laboratory abnormalities, the studies were repeated with greater frequency. After release from the isolation ward, the hepatic tests were continued at 1- to 3-week intervals.
Inoculation of volunteers. On each of 4 successive days,
5 ml. of feces pool 13-14-15 FIH was administered orally
in chocolate milk to each of 5 volunteers. Each of 5
other volunteers ingested 3 ml. (1 man) to 5 ml. (4 men)
TABLE I

Results in volunteers afkr ingestion of
feces pool 13-14-15 FIH

Sub-

Age

J. D.

31

ject

R. A.

G. S.
E. S.
A. M.

IUnless
(-to

D. A. I.

Remarks

Symptoms only
* +++ 127 to 137 Symptoms, liver tenderness, mild hepatic
________ dysfunction
32
+
18 to 41 Intermittent symp+

20 to 28

toms

20 to 35

27

+
-

33

-

-

23

Symptoms only

-

Illnesses graded from -(no illness) to + + + + on the
basis of the relative severity of symptoms and signs as observed in these subjects. D.A.I. indicates the number of
days after inoculation when symptoms were present.
TABLE II

Results in volunteers after ingestion of
liver suspension 1-CLIH
Sub-

jects

Age

E. Mc. 29

Illness

(- to

D. A. I.

Remarks

+

22 to 29

++++

36 to 50

Symptoms
Symptoms, plus hepatic enlargement and
tenderness; no significant hepatic dysfunction detected

T. K.

21

+

26 to 30

M. P.

28

+

28 to 51

P. S.

34

+

15 to 60

J. M.

34

-

Symptoms and laboratory evidence suggestive of mild hepatic dysfunction
Intermittent symptoms only
Intermittent symptoms only

See legend of Table I for other data referable to interpretation of table.

334

JOHN R. NEEFE, JOSEPH STOKES, JR., ROBERT S. GARBER, AND SYDNEY S. GELLIS
TABLE III

Results in volunteers after ingestion of
serum pool 1-CSIH
Sub

jects

Age
b-Illness
(- to
+

D. A. I.

Remarks

Very mild symptoms
only
Very mild symptoms
only

)

V. P.

39

4

10 to 15

E. S.

31

4-

14 to 26

W. H. 32

-

_

_

A. W.

20

-

_

_

L. S.

29

See legend of Table I for other data referable to interpretation of table.

of the undiluted serum pool 1-CSIH, the material being
administered in milk. The 5 men of the third group each
ingested (in milk) 20 ml. of liver suspension 1-CLIH.
Previous experience with the strain of infectious hepatitis virps (virus IH) that was used for induction of
hepatitis in the three volunteers from which the present
materials were obtained had shown it to be highly effective in inducing the apparent disease when administered
orally but much less effective when administred parenterally. For this reason, and because of the limited number
of volunteers available for these preliminary studies, only
the oral route of inoculation was used.
RESULTS

The results obtained from the, oral administration of the feces, liver, and serum preparations are
sununarized in Tables I, II, and III and are described in detail below.
Feces pool 13-14-15 FIH
J. D. (AGE 32). Eleven days after inoculation
he complained of cramps in the abdomen. On the
16th day, he noted a "slight rash" associated with
pruritis. As this was transient, it was not observed by a physician and a reliable description is
not available. From the 20th to 28th days inclusive, he complained of nausea, headache, and
general malaise. No significant physical findings
were recorded during this period, and no definite
evidence of hepatic disturbance was revealed by
the various hepatic tests. After the 28th day, he
apparently was well until the 127th day. He had
been released from the isolation ward and returned to the New Jersey State Prison on the
70th day. On approximately the 122nd day, he

was released from the prison and went to his
home. From the 122nd to 127th days, he had
little sleep and ingested moderate quantities of
alcoholic beverages. On the 127th day, he noted
extreme malaise. This was associated with chilly
sensations and his temperature was found to be
102.80 F. (oral). He was admitted the next day
(128th) to the Hospital of the University of
Pennsylvania. At that time, he complained chiefly
of weakness, headache, aching of the eyes on
movement, anorexia, and pain in the right costovertebral angle. Examination revealed an oral
temperature of 1020 F., slight enlargement of the
inguinal, axillary, and cervical lymph nodes, and
pronounced tenderness in the right upper quadrant and right costovertebral angle. The liver
and spleen were not palpable. The symptoms and
findings persisted until the 132nd day after which
they gradually subsided. By the 137th day, he
was asymptomatic and physical examination was
negative. The oral temperature varied between
990 and 1010 F. on the 129th and 130th days,
from normal to 100.40 F. on the 131st and
132nd days, and thereafter was normal. He was
discharged from the hospital on the 137th day.
The results of the laboratory studies made during this period were as follows: The blood hemoglobin varied between 90 and 95 per cent; the
total leukocyte count was 5,000 on the 128th day
and 5,000 on the 131st day. The differential
leukocyte count on the 131st day revealed: neutrophiles-59 per cent, lymphocytes-40 per cent,
monocytes-1 per cent. Routine urinalysis consistently revealed no abnormal findings. The
hepatic tests gave the following results suggesting
the existence of mild hepatic disturbance: The
Harrison spot test for urine bilirubin was constantly positive between the 128th and 139th days,
the response varying from 1 to 3 +. Urine urobilinogen was somewhat increased, the maximum
recorded value being 2.0 Ehrlich units in a morning,2-hour specimen. Bromsulphalein tests on the
128th, 129th, 130th and 137th days gave 30- and
45-minute values (30 minute per cent retention
-45 minute per cent retention) of 7/4, 12/11,
7/4, and 4/3 respectively. The results of the
total and prompt direct reacting serum bilirubin
determinations, the cephalin-cholesterol flocculation, collodial gold and thymol, and the serum pro-

STUDIES ON THE HEPATITIS VIRUS

tein and cholesterol studies showed no significant
variations from the normal.
Typhoid H and 0, paratyphoid A and B, and
brucella abortus agglutinations on the 126th day
were negative. A heterophile antibody test (PaulBunnell) on the 137th day (maximal response
graded 4 +) gave 2 +, 1 +, and negative reactions in the 1 :32, 1 :64, and higher dilutions respectively.
Although very cooperative, this subject was
anxious to leave the hospital as soon as possible
due to the fact that he had been released from
prison just prior to onset of this illness. As he
felt perfectly well by the 137th day, it was not
possible to detain him for further studies after
that time. Two months later, he returned for
follow-up studies, having been entirely well during the interval. All hepatic tests were normal
and the heterophile antibody test gave a 1 + reaction in the 1: 64 dilution, the reaction being
negative in the higher dilutions.
R. A. (AGE 32). Eighteen days after inoculation,
this subject complained of general malaise and
moderate pain in the lumbar area of the back.
Between the 19th and 22nd days, he voluntarily
remained in bed and complained of malaise, backache, and anorexia. On the 23rd day, he had intermittent chilly sensations during the evening. These
continued during the 24th and 25th days and were
associated with nausea and one episode of vomiting. Although he then felt relatively normal until
the 39th day, his oral temperature varied from
99.80 to 100.80 F. between the 34th and 37th days.
From the 39th to the 41st day, he complained of
persistent headache and intermittent nausea.
After the 41st day, he remained asymptomatic.
No significant physical findings, other than the
slight elevation of temperature, were recorded.
The hepatic studies revealed no definite evidence
of hepatic disturbance. However, the thymol test,
which had not given 30-minute turbidity readings
greater than 1.5 units or 18-hour flocculation reactions greater than 1 + with numerous serum
specimens obtained during the month prior to inoculation, gave turbidity readings up to 3.0 units
and 2 + flocculation reactions during the period
of symptoms.
G. S. (AGE 23). On the 11th day, the subject
complained of slight nausea, headache, and abdominal cramps that persisted for only 24 hours.

335

From the 20th to the 35th days, he had frequent
headaches, intermittent lumbar backache, and mild
to moderate anorexia and nausea. On the 25th
and 30th days, he vomited several times. The only
laboratory abnormality was the finding of an increased excretion of urine urobilinogen (2.0 E.
U.) on the 34th day. He remained well after the
35th day.
SUBJECTS E. S. (AGE 27) AND A. M. (AGE 33).
The remaining two volunteers who ingested feces
pool 13-14-15 FIH experienced no significant
symptoms, and no evidence of hepatic disturbance
was revealed by hepatic tests during the period of
observation.
Liver suspension (1-CLIH)
E. MC. (AGE 29). This subject was well until
the 22nd day when, for a period of 24 hours, he
reported general malaise, anorexia, nausea, and
one episode of vomiting. Although he was relatively asymptomatic between the 23rd and 26th
days, symptoms recurred on the 26th day, and persisted till the 29th day. During this period he
complained of weakness, general malaise, anorexia,
nausea, and pain in the region of the right costovertebral angle. He was relatively free from symptoms between the 29th and 36th days. However,
from the 36th to the 50th day, he experienced
rather severe symptoms. They included: (1)
marked malaise of sufficient intensity to keep him
in bed; (2) complete anorexia, severe nausea, and
vomiting one or more times daily for a period of
approximately 10 days; any attempt to eat solid
food was followed by vomiting; (3) headache
with considerable orbital pain on movement of
the eyes; (4) pain in the right upper quadrant that
at times was of sufficient severity to require morphine. He was given daily intravenous infusions
of glucose and saline during this period. No
significant temperature elevations were recorded
until the 48th day when the oral temperature was
1000 F. It fluctuated between 980 and 1000 F.
on the 48th and 49th days. About 2 hours after
a plasma transfusion on the 50th day, he had a
chill and, during the next few hours, his temperature gradually rose to a maximum of 1070 F.
(rectal). The significance of the lever noted on
the 48th and 49th days is uncertain as he had received glucose and saline infusions on these days.
That of the 50th day appeared to be the result of

336

JOHN R. NEEFE, JOSEPH STOKES, JR., ROBERT S. GARBER, AND SYDNEY S. GELLIS

a plasma reaction. With the subsidence of this
reaction, all of his symptoms diminished rapidly
and he felt relatively normal by the 65th day.
Physical examination throughout the period of
severe symptoms (36th to 50th days) revealed
marked tenderness in the right upper quadrant.
The liver was enlarged and quite tender. With
inspiration, the edge of the liver was easily palpable 3 to 4 cm. below the costal margin. After
subsidence of the symptoms, the size of the liver
decreased. The spleen was not palpable. No
other significant physical abnormalities were detected.
The several blood counts (hemoglobin, total and
differential leukocyte counts) made during the
period of acute symptoms revealed no significant
abnormalities. Hepatic tests revealed no significant abnormalities until the 49th day when the
urine gave a positive reaction for urobilinogen in
a dilution of 1 to 30 and bromsulphalein retention
was 10 per cent at 30 minutes and 3 per cent at
45 minutes.4 The following day the 30- and 45minute retentions were 40 and 30 per cent respectively. The urine was positive for urobilinogen in dilutions of 1 to 100 and 1 to 150 on the
50th and 51st days. On the 55th day, difficulty
was encountered in obtaining a 30-minute blood
specimen for the bromsulphalein test. A specimen obtained at 41 minutes revealed 8 per cent
retention. The other hepatic tests revealed no
significant abnormalities and thereafter all results were within normal limits. The abnormalities mentioned are of doubtful significance in respect to his illness because of their relationship to
the reactions from the intravenous infusions.
Roentgenological examination of the gastrointestinal tract on the 57th day revealed no abnormal
findings.
T. K. (AGE 21). Between the 26th and 30th
days after inoculation, he complained of malaise,
mild anorexia, nausea, one episode of vomiting,
and headache. These subsided by the 30th day
and he remained well thereafter. There were no
abnormal temperature elevations.
The hepatic tests during this period revealed a
maximum total serum bilirubin of 1.7 mgm. per
4With 7 previous bromsulphalein tests performed during the preceding 2 months, retention at 30 and 45 minutes
was never greater than trace and 0 respectively.

100 ml. (pre-inoculation maximum 1.2 mgm.),
a delayed direct qualitative van den Bergh reaction, a maximum thymol turbidity of 4.0 units
(maximum pre-inoculation reading-2.5 units),
and a maximum 2-hour urine urobilinogen concentration of 6.0 Ehrlich units (maximum preinoculation 1.6) with a positive reaction also being obtained in a urine dilution of 1 to 200.
J. M. (AGE 34). This subject had no significant
symptoms or laboratory manifestations of hepatic
disturbance following inoculation.
M. P. (AGE 28). Between the 28th and 51st
days following inoculation, he complained of
malaise and intermittent rather severe headaches.
No other significant symptoms or laboratory
manifestations suggestive of hepatic disturbance
were present.
P. S. (AGE 34). Fifteen days after inoculation
a pruritic rash, slight headache, and an elevation
of oral temperature to 1000 F. were experienced.
No adequate description of the rash is available
as it was observed only by the attending nurse and
disappeared in 24 hours before he was seen by a
physician. From the 21st to 26th days he was
nauseated intermittently, vomited on two occasions, and his appetite was moderately diminished.
He then was relatively asymptomatic until the
39th day when anorexia, abdominal cramps, and
headache were present and persisted for approximately 2 days. He again was free from symptoms
until the 59th day when, for a 24-hour period, he
had anorexia, nausea, and one episode of vomiting.
Thereafter he remained well. No significant laboratory evidences suggestive of hepatic disturbance
were detected at any time.
Serum pool (1-CSIH)
None of the 5 men who ingested this serum (V.
R., age 39; E. S., 31; W. H., 32; L. S., 29;
A. W., 20) developed significant laboratory evidence suggestive of hepatic disturbance following
their inoculation. One (V. P.) complained of
slight nausea and mild headache between the 10th
and 15th days. Another (E. S.) between the 14th
and 26th days, had malaise, and intermittent mild
nausea and headache. The symptoms in both
were not sufficient to interfere with their voluntary continuance of their usual activities.

STUDIES ON THE HEPATITIS VIRUS
DISCUSSION

The results of the present studies permit no
definite conclusions concerning the presence of a
hepatitis virus in the serum, feces, and liver preparations. Nevertheless, four of the five men who
ingested the liver suspension and three of the five
who ingested the feces preparation developed illnesses, of mild to moderate severity, after intervals corresponding to the usual incubation period
of infectious hepatitis. The symptoms of subject E. McE. (liver suspension) were typical of
infectious hepatitis of moderate severity and were
associated with enlargement and tenderness of the
liver. The only definite laboratory evidence of
hepatic disturbance in this subject was observed
late in the course of the illness. This was quite
transient and could be explained entirely on the
basis of febrile reactions that followed intravenous
glucose, saline, and plasma. Except for the lack
of laboratory evidence of hepatic disturbance, his
illness clinically was typical of infectious hepatitis
without jaundice.
The symptoms and physical findings in volunteer J. D. (feces preparation) beginning 127 days
after inoculation also were typical of mild infectious hepatitis without jaundice. Liver tenderness
and laboratory evidence of mild hepatic disturbance were present. This illness began after a
period of unaccustomed physical activity, inadequate rest, and ingestion of considerable
amounts of alcohol, all following his release after
a number of years in prison. Although the possibility that this illness was coincidental and unrelated to the experiment obviously cannot be excluded, it also is quite possible that his symptoms
beginning on the 20th day were the result of the
hepatitis virus and that his resistance was sufficient to protect him until it was lowered by the
activities following his release from the prison.
Such activities have been related to an increased
incidence of relapse in infectious hepatitis.
The illnesses of the other men were mild. The
symptoms and the interval between inoculation and
their onset were similar to the symptoms and incubation period of infectious hepatitis. However,
evidences of hepatic disturbance either were absent or minimal. In other volunteers with symptoms of comparable and even less severity due to
hepatitis resulting from inoculation with this strain

337

of virus, as obtained from acute cases, the illness
usually has been associated with fever, and hepatic
tests consistently have revealed evidence of marked
hepatic disturbance. If these illnesses are considered to be due to a hepatitis virus in the materials obtained from the volunteers with chronic
hepatitis, the difference in the manifestations observed in these volunteers from those observed
in others inoculated with materials from acute
cases must be explained. Some change in the
virus itself, a difference in the resistance of the
volunteers used, or both possibly could acc6unt
for the difference. Unfortunately, the volunteers
available at the time of the present experiment did
not include persons in the age groups most susceptible to the strain of virus concerned. Previous
studies (2) have shown that the age range of greatest susceptibility to this strain of virus was from 6 to
25, the incidence in exposed persons over this age
being much lower. Furthermore, with the more
marked decrease in incidence observed after the
age of 30, the incidence of hepatitis without jaundice in those over this age who acquired the disease increased from 26 per cent in the 10 to 30 age
group to 66 per cent of those over 30. Of the 15
volunteers available for the present study, only 3
(1 in each group of five) were under 25, 4 were
between 25 and 30, and 8 were over 30 years of
age. It is possible, therefore that the illness observed in these volunteers represented the response
of relatively resistant hosts to the hepatitis virus.
On the other hand, it also is possible that, in the
volunteers with chronic non-icteric hepatitis, some
modification of the virus had occurred. However,
lacking any specific serological or other test for
the hepatitis virus, conclusions regarding the significance of the present results must be deferred.
It is hoped that additional volunteers will become
available for further tests of the materials used in
the present study and of materials obtained from
the present subjects during their etiologically obscure illnesses. In addition to the possible influence of factors mentioned above, the failure of
serum pool 1-CSIH to produce any significant
manifestations suggesting the presence of the hepatitis virus also could have been due either to the
absence of the virus in the pool or to the presence,
in some of the specimens, of neutralizing substances which inactivated the virus. Thus, the

338

JOHN R. NEEFE, JOSEPH STOKES, JR., ROBERT S. GARtER, AND SYDNEY S. GELLIS

negative results with this serum pool do not necessarily indicate that the virus was absent from all
of the individual specimens.
SUMMARY AND CONCLUSIONS

Serum and feces specimens and liver tissue (biopsy) were obtained from 3 subjects who had persistent symptoms and other evidence of continued
hepatic disturbance (chronic nonicteric hepatitis)
following acute hepatitis that had been induced by
oral inoculation with a known strain of "infectious
hepatitis" virus. In an attempt to clarify the relationship of the hepatitis virus to the persistent
symptoms and continued hepatic disturbance, volunteers were inoculated orally with materials collected from 3 to 12 months after the onset of the
disease. Following ingestion of the feces preparation, 3 of the 5 volunteers developed mild illnesses
after 18 to 20 days. Likewise, 4 of the 5 volunteers developed illnesses of mild to moderate
severity 15 to 36 days after ingestion of a suspension of the liver tissue. None of the 5 volunteers
who ingested the serum pool developed significant
illness during a 5-month period of observation.
The symptoms and manifestations of those who became ill included two or more of the following:
malaise, headache, anorexia, nausea, vomiting, abdominal cramps, and pain in the right upper quadrant. Two presented tenderness in the liver area
and, in one of these, the liver became moderately
enlarged.
In spite of moderately severe symptoms in two
of the cases, laboratory evidences of hepatic disturbance in all cases either were absent or minimal.
None developed overt jaundice. The intervals between inoculation and onset and the clinical manifestations were compatible with mild infectious
hepatitis without jaundice, particularly in two of
the cases. The laboratory evidences of hepatic
disturbance usually associated with mild hepatitis,
however, were not present. Although no other ex-

planation for the illnesses is apparent, their exact
nature and their relationship to the materials used
for inoculation are uncertain. Factors of possible
importance in interpretation of the results are discussed.
ACKNOWLEDGMENT

The authors wish to express their appreciation and
gratitude for the contribution of those inmates of the
New Jersey State Prison, Trenton, N. J., who volunteered as subjects for the investigation. The assistance
and cooperation of the administrative staffs of the Department of Institutions and Agencies, State of New Jersey, the New Jersey State Prison, and the New Jersey
State Hospital, Trenton, N. J., also are gratefully acknowledged as is the technical assistance of Mr. and Mrs.
Charles Ming and -Mr. Ewing Joiner in the conduct of
the laboratory studies.

1.
2.
3.

4.

5.

6.

7.

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