野口英世著

Journal of Experimental Medicine

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A FILTER-PASSING VIRUS OBTAINED FROM DERMACENTOR ANDERSONI.
BY HIDEYO NOGUCHI, M.D.
(From the Laboratories of The Roclefeller Institute for Medical Research.)(Received for publication, February 17, 1926.)

In the course of a bacteriological study of Dermacentor andersoni,
the wood tick concerned with the transmission of Rocky Mountainspotted fever~ and of tularemia, ~ a filter-passing virus was met with
and, apparently, carried through seven generations in vitro, as well as
through experimental animals. The history of the tick material
with which these studies were made is as follows:

Source of the Virus.
The tick from which the virus was isolated was one of a lot of 51"drag" ticks collected in Saw Tooth Canyon, Montana, in the springof 1925 and sent me through the courtesy of Dr. R. R. Parker, of theUnited States Public Health Service. Four ticks of this lot, after
having been kept in the refrigerator (10-12°C.) for 54 days, wereplaced for 48 hours at 37°C. preparatory to tests for infection withspotted fever virus, according to the procedure of Spencer and Parker.*
The tests were first made by allowing the ticks to feed for 72 hours,
each on a normal guinea pig. Ticks 1, 3, and 4 became engorged withblood; Tick 2 was found dead. The guinea pig on which Tick 1 had
fed developed typical spotted fever, the others remained well. The
ticks were then eviscerated, the viscera were individually suspended
in 4 cc. of salt solution, and 1 cc. of each suspension was injected into
a normal guinea pig. The suspensions of Ticks 1 and 2 induced
typical spotted fever, that of Tick 4 produced no reaction. The susJ Ricketts, H. T., J. Infect. Dis., 1907, iv, 141.
2 Francis, E., J. Am. Med. Assn., 1925, lxxxiv, 1243.
3Spencer, R. R., and Parker, R. R., Pub. Health Rep., U. S. P. tt., 1924,
xxxix, 3027.
1

VIRUS FROM

DERMACENTORANDERSONI

pension of Tick 3, which contained an unusually large number of microorganisms very similar to those found in infected ticks, induced afever resembling spotted fever but one which conferred no immunity
to the spotted fever virus. It is this infective agent existing in Tick
3 with which the present report is concerned.
Simultaneously with the injections into guinea pigs, each tick suspension was inoculated into special culture media, which are describedin another paper? and duplicate series of tubes were kept at 26°C.
and at 37°C. The presence of ordinary bacterial contamination was
TABLE
Tick No.

Result of feeding.

Typical spotted fever.

2(founddead).

I.

(Perhaps never attached.)

No immunity developed.

m

No immunity developed.

Result of inoculation.

Suspension (containing moderate number oforganisms similar to those in suspension ofTick 3) induced typical spotted fever,Suspension (containing some microorganisms)
induced typical spotted fever.

Suspension (containing unusually large numbers of microorganisms) induced feververy similar to that of spotted fever but
none of the skin lesions of the disease.
Immunity test, 17 days after inoculation,negative.
Suspension (in which no microorganisms could
be demonstrated) produced no reaction.
No immunity developed.

controlled by the use of plain broth and agar slants. The culture
made with the suspension of the dead tick, Tick 2, was discarded
because of mold contamination. Cultures made from the suspensions of Ticks 1, 3, and 4 were tested on guinea pigs at various intervals, beginning as early as 72 hours of incubation. The amount of
the culture used for inoculation was 0.1 cc. or less and was usuallysuspended for convenience in 1 to 2 cc. of saline.
No fever or other reaction developed in guinea pigs which had
4 Noguchi, H., J. Exp. Med., 1926, xliH, 515.

nLu~.¥O NOGUCHI

3

received the 72 hour cultures of Ticks 1 and 4. When tested withspotted fever virus 18 days after the inoculation, the animals inoculated with the Tick 4 cultures developed spotted fever, those which hadreceived Tick 1 cultures proved immune. No real culture, however,
was obtained with the suspension of Tick 1; it was completely inert
when tested again 25 days later.
The cultures made with the suspension of Tick 3, however, gave
rise to exactly the same type of fever in guinea pigs as had the original material. The 72 hour cultures proved active, and as time wenton their activity gradually increased. As no microorganisms could be
detected by dark-field examination or by any staining method, theinfective agent present in the culture will be referred to here as avirus.
The virus is aerobic; it is capable of growing at 26°C. and 37°C.
and has been continued in subcultures for seven generations at 37°C.
It does not grow in ordinary broth or on slant agar. It produces aslight turbidity when grown in serum media containing certain carbohydrates (mixed sugars). No putrefactive odor or gas is produced.
The blood and spleen,--the latter organ always much enlarged, dark
red, and soft,--of guinea pigs responding to the cultures of the virus
are infective, and continuous passages from animal to animal up to the
7th generation have been maintained with the material taken on the
3rd, 4th, and 5th days of fever. No microorganisms have been seenin cultures made with the blood or spleen of such animals. The virus
was recovered from the blood of infected guinea pigs. It proved to be
filterable through Berkefeld N filters.
Chart 1 shows the type of temperature curve observed in infectionof guinea pigs with the virus under discussion. The result of an
immunity test with spotted fever blood after recovery is also indicatedin the chart.
EXPEI~'rMF-NTAL.

Original Material.
B-C Tick 3, fed for 72 hours on guinea pig infected with spotted fever virus,
Barlow-Cooper strain. 54 days later, fed on normal guinea pig 72 hours. Resultof feeding: Animal showed no sign of infection. Result of inoculation of suspended
viscera: Fever (Chart 1) developed 72 hours after inoculation; animal remainedVIRUS ~ROM DERMACENTOR ANDERSONI

febrile7 days. Blood withdrawn at the height of fever sterile. No scrotal or
ear lesions appeared, and subsequent inoculation with spotted fever virus (17days after inoculation of tick suspension) induced the typical symptoms andlesionsof the disease.

Culture Experiments.
1st generation.--A normal guinea pig received 0.1 cc. of culture (special semisolid and slant media) kept 3 days at 26°C. after inoculation with 0.1 cc. of suspension of Tick 3. After 4 days the animal developed high temperature which
continued for 4 days (105, 106, 104.5, 104.4°F.). No scrotal lesions appeared.
Tested for immunity by injection of spotted fever virus (guinea pig blood)after
23 days. No protection.
~yof

dls~

Tickvi~ m~ui~ion
I 2

5 4

~o8~
L~ _

~0~ ~

Test with spotte¢lfev¢~l~us

5 6 7 8 g I0 n 12 15 14 15 16 17 l ~ I g 2 0 21 ~ ~ 4 2 5

~'~
_ _ _

~ ~. l ~

.~.

r ~

~ ~

~

104

Z627 2 8 2 9 ~ 0

~

/

.

~

- ~~" --

Cm~mT 1.

Similar material, kept 3 days at 37°C., induced fever after 3 days incubation,
which lasted 6 days (104.4, 105.6, 105, 103.8, 104.4, 104.2°F.). No scrotal lesionsat any time. No immunity to spotted fever virus, as shown by immunity test
31 days after culture inoculation.
The same material, after 10 days at 37°C., induced a similar febrile reaction in
two guinea pigs. In one, fever appeared after 4 days and lasted 6 days (104.6,
105.6, 105.6, 105.4, 105.2, 104.6°F.). No scrotal lesion developed. The animal
showed no immunity against spotted fever when tested 12 days later. The
second animal reacted with fever after 4 days and was killed for examination on
the 5th day of fever (104.4, 105.2, 105.6, 106, 105°). The spleen was greatly enlarged and dark red, not unlike the spleen of a guinea pig infected with spotted
fever; but no other lesions were found.
After 13 days at 37°C., the same material induced after 3 days incubation a

H~VvYO NOGUCHI

5

fever lasting 7 days. No scrotal lesions developed. When tested with spotted
fever virus, 15 days after the inoculation of culture, this animal escaped infection.
Two other guinea pigs which were inoculated with the same culture material andalso reacted with fever did not prove to be immune to the spotted fever virus.
After 17 days at 37°C., the material was tested for its virulence by injecting
guinea pigs with dilutions, as shown in Table II.
A second set of cultures was made with the same suspension after it had been
kept 10 days at 0°C., and the culture tubes were placed at 37°C. for 8 days. A
guinea pig which received 0.1 cc. of culture developed high temperature after 3
days incubation and remained continuously febrile for 8 days (104.6, 106.4, 106,105.2, 105.6, 105, 104.8, 104.2°F.). No scrotal lesions developed at any time.
2nd generation.--Subcultures, made on the same media as the original cultures,and kept for 10 days at 37°C., were injected into two guinea pigs. High temperature developed in both animals after 6 days and was continuous in one for 6 days
(104.4, 104.4, 104.6, 104.8, 104.4, 104°F.) and in the other for 5 days (105, 105.4,
TABLE II.
Amount injected.

Incubation period.

Duration of fever.

f,¢oImmunit~ tests.with spottedlev¢~ vlrus.

days

d~ys

1

3
4

7
7

No pmtectlon.

0.1
0.1
0.01
0.001

3

8

4

7

Test not made.
No protection.

0.00015

4(105.4,105.6, 104,104.8eF.)
7(Chart 2.)

tc~g

Test not made.

105.2, 104.6, 104°). No scrotal lesions appeared. The first animal was tested for
immunity to the virus 16 days after the culture inoculation. It proved immune.
The second was tested with spotted fever virus after the same period. It reacted
typically.
A second set of subcultures was kept 9 days at 37°C. and tested in normal
guinea pigs. The animals had high temperature after 5 days which continued
for 6 days (104, 104, 105.4, 105.8, 106, 104°F.). No scrotal lesions developed, andno protection against the spotted fever virus could be demonstrated. After 13days at 37°C., the material gave rise after 3 days to high fever which lasted 7 days
(104, 104, 105.5, 106.8, 107, 105.5, 104.5°), but no scrotal lesions appeared(Chart 3).
3rd generation.--Subcultures from tubes of the 2nd generation, kept 4 days at
37°C., induced in guinea pigs, after 6 days incubation, a fever lasting 5 days
(104.5, 106.5, 106, 105.5, 105°F.) (Chart 4). There were no scrotal lesions.
Another set of subcultures, after 8 days at 37°C., induced mild fever after 10 days
incubation.

5

VIRUS ~ROM DEI~.ACENTOR AND]~RSONIu~!i_t,.'l I I
II~ll t I I'.,J

I
I

ltlll~

I

,,,-4

•

0

0

0

0

0

0

0

0

0

HIDEYO NOGUCHI7

I n C h a r t s 2, 3, a n d 4 are presented t e m p e r a t u r e curves of some of
the guinea pigs inoculated w i t h cultures of the 1st, 2nd, a n d 3rd
generations.

4th generation cultures, 7 days old at 37°C., induced in one guinea pig after 10days incubation a fever lasting 5 days, and in another fever of 3 days duration.
5tk generation.--7 day old cultures, kept at 37°C., induced a febrile reaction
lasting 4 days, the incubation period being 10 days.
6tk generation cultures, 10 days old at 37°C., induced fever of 5 days duration
after an incubation period of 7 days.
7th generation.mSubcultures from the 6th generation, after 4 days at 370C.,
induced fever of 5 days duration following an incubation period of 10 days.
The number of positive reactions became less constant with further subcultures, the virus becoming gradually attenuated.
Macacus rhesus/.--The monkey received an intraperitoneal injection of 1 cc.
of mixed cultures of the virus, including one of the 1st generation (28 days old)and one of the 4th (8 days old). A fever fluctuating between 104°F. and 104.8°
developed after 3 days and lasted 6 days. No spotted fever lesions appearedin the animal.
Guinea Pig Passages.
1st passage.--Guinea Pig XVIII-84B developed fever (104-I05.5°F.) 5 days
after receiving a 1st generation culture (13 days old at 37°C.), the temperatures
recorded on successive days being 105.2, 105.4, 106, 105.50F. The animal waS
killed on the 5th day of fever. The spleen was enlarged, and its appearance
suggested that of the spleen in spotted fever infection. The blood, which was freefrom contaminating microorganisms, as tested by cultures, was injected intraperitoneaUy into two normal guinea pigs.
2nd passage.--Guinea Pig XVIII-121A5 developed fever after 4 days which
continued for 7 days (106.2, 105.4, 104.6, 104°F.). Guinea Pig XVIII-121B
also showed high fever after 4 days (105.6, 106.4, 105.4, 105.4°F.). It was killedon the 4th day of fever for passage, cultural experiments, and virulence tests onmonkeys, rabbits, and guinea pigs.
3rd passage.uCitrate blood from the animal just mentioned was injected into
four guinea pigs, the first receiving 1 cc., the second 0.1 cc., the third 0.01 cc.,and the fourth 1 cc. The fourth guinea pig received in addition 2 cc. of serum fromrabbits immunized to spotted fever, of a titre such that 0.1 cc. neutralized 100~.T..D. of spotted fever virus (guinea pig blood). The incubation period in each
instance was 7 days, and the duration of fever was 2 to 3 days in the first three
animals and 6 days in the fourth. There was no evidence of any neutralizing
effect on the virus by spotted fever antiserum.
5 Twenty-two ticks were placed on this animal on the 3rd day of fever andallowed to feed 5 days; nineteen became engorged, three were found dead.

8

VIRUS ]FROM DERMACENTOR ANDERSONIThe same blood was injected into Macacus rhesus 2 and a rabbit intraperitoneally, each receiving 2 cc., but no reaction followed in either instance.
After the 3rd passage, successive transfers were made as routine, two guinea
pigs being used in each passage, with two sets oi cultures set up for each, until the
8th passage, 3 months after the 1st, when the work was discontinued. No increasein virulence was noted, but on the contrary the duration of fever tended to become
shorter and the incubation period longer. At each passage the blood was tested
for ordinary bacterial invasion, as for example by bacilli of the paratyphoid group;
no such invasion was discovered.

Filtration Experiments.
Cultures of the 1st, 2nd, and 3rd generations, grown on the special semisolidand slant media, were pooled and diluted with an equal volume of 0.9 per cent salt
solution. The suspension, which contained no demonstrable microorganisms,
was thoroughly shaken and passed through a Berkefeld filter N. The clear, lightbrownish filtrate was injected into four guinea pigs in amounts of 4, 3, 2, and 1 cc.,
respectively. The first three animals developed, after 4 days incubation, typical
fever (104.5-105.5°F.) which was continuous for 7 days; the guinea pig which was
inoculated with 1 cc. showed slight fever for 2 days. These animals were tested
for immunity by injections of virulent spotted fever blood, but all had typical
severe infections.
A guinea pig which had been injected as control with 1 cc. of the unfilteredsuspension became ill in 4 days and passed through a course of fever lasting 5 days
(106.2, 105.6, 106, 104.6, 104.6°F.).
Subcultures made with the filtrate yielded an active culture, which was subsequently tested for filterability, with positive results. Neither filtrate nor cultures contained any demonstrable microorganisms.
T h e question arose whether the infective agent revealed b y the
inoculation of guinea pigs had not merely survived in the culture
tubes a t 37°C. T h e original suspension, preserved a t 3°C., wastested in guinea pigs in various dilutions on the 7th and 17th days
after its preparation. T h e results, which are recorded in Table III,
show t h a t 1 cc. of the original material induced infection, but not 0.1
cc. or less. T h e culture of the 1st generation, on the other hand, when
17 days old was effective in a dose of 0.0001 cc.
As has already been mentioned, the original suspension contained
an enormous n u m b e r of minute, non-motile, Gram-negative microorganisms resembling spotted fever organisms. These did not growon the media employed for cultivating the filterable virus underdiscussion.

HIDEYO NOGUCHI

9

Transmission of the Virus from Infected Guinea Pigs to Ticks andVice Versa.
Experiment /.--Seven ticks which had been fed for 5 days on a guinea pig
in the febrile stage of the virus infection were placed separately on seven normal
guinea pigs and allowed to feed for 5 days. In one of the animals fever developed
after 8 days (104--105°F.) and lasted 4 days. The others escaped infection.
The tick which had induced infection was killed 119 days later and a portior*of the emulsified viscera inoculated into a guinea pig. Fever (I04.5-105.5°F.)
developed after 48 hours and lasted 8 days. Death occurred on the llth day.
There were no lesions of spotted fever. The spleen was soft and considerably
enlarged. No bacteria could be demonstrated in cultures made from the bloodand spleen. Inoculation of blood withdrawn during the febrile stage induced theusual prolonged febrile, non-fatal reaction in guinea pigs; no spotted fever lesions
developed.
TABLE III.
Amount of tick suspension.

1

0.1
0.01
0.001

Kept 7 days at 3°C.

Kept 17 days at 3~C.

Usual fever.
No reaction.

Usual fever.
No reaction.

Smears of the tick revealed only a few microorganisms, but sections showed alarge number of minute Gram-negative elements in the lumen of the intestine aswell as in the viscera. Cultures made with the suspension yielded no growth.
Experiment 2.--A female tick, which had failed to induce infection by biting,survived ll0 days and laid several lots of eggs. The tick and 40 of the eggs wereseparately suspended in saline, and a portion of each was injected into guinea
pigs. No fever developed. In the smears and sections of the tick and eggs were
found a number of Gram-negative microorganisms, and cultures yielded a growthof B. ric~ettsiformisJ The finding was of interest in showing hereditary transmission of this non-pathogenic microorganism, which had already been found in
the ovaries and egg cells of ticks.
Experiment 3.--Four ticks of a lot of eleven which had been fed for 5 days on aguinea pig infected experimentally were still living after 124 days and were separately fed on guinea pigs, but no fever was induced. The remaining seven ticks
were dead but only partly dried, and a single suspension in salt solution was madeof their viscera. The guinea pig injected with a portion of this suspension developed after 8 days incubation a fever of 5 days duration. The blood withdrawn
on the 4th day of fever contained no contaminating bacteria. The filtrate of theemulsion failed to induce infection.

10

V I R U S ~'ROM" D E R M A C E N T O R

ANDERSON][

This survival of the virus in ticks for several months at refrigerator
temperature is not surprising, since it occurred also in culture. A
guinea pig receiving 0.2 cc. of 1st generation culture of the virus,
preserved at 2°C. for 5 months, developed the typical febrile reaction
after 4 days and remained febrile for 6 days, when it was killed fortransfer and culture. There was no bacterial invasion.
The foregoing experiments show that the virus is taken up by ticks
from the blood of the infected guinea pig, but that transmission of theinfective agent to other guinea pigs by the bite of infected ticks seldom
occurs under the conditions described. The presence of the virus inticks can be demonstrated more definitely by inoculation of guinea
pigs with tick emulsions. That all ticks which are fed on infectedguinea pigs do not take up the virus, however, is shown by the negative
results in some instances.

Cross-Neutralization Experiments.
It has been repeatedly noted that guinea pigs which have responded
to the virus under discussion do not acquire immunity against the
spotted fever virus. Conversely, guinea pigs which have passed
through the spotted fever infection were found to be quite as susceptible to this virus as normal animals. A potent spotted fever immune
serum .failed to neutralize the cultured virus.
SUMMARY.

An invisible, filter-passing virus, pathogenic for the guinea pig, andcapable of cultivation on special media for at least seven generations,has been isolated from a tick of the species Dermacentor andersoni.
One of two monkeys (Macacus rhesus) inoculated became infected, andin one rabbit the result was negative.
The virus has been transmitted from infected guinea pigs to ticks
as also in one instance by tick feeding from an infected tick to a guinea
pig The presence of the virus in the tick is more easily demonstrated
by the inoculation of guinea pigs with a suspension of the tick viscera.
Continuous high fever (104.5-106.5°F.) and enlargement of the spleen
are the chief symptoms of the infection in guinea pigs. After the
febrile attack the guinea pigs are not susceptible to reinoculation withthe cultured virus but are subject to infection with the virus of spotted
fever, and vice versa.