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u. s. ARMY CHEMICAL CORPS RESEARCH AND DEVELOPMENT COMMAND
U.S. ARMY BIOLOGICAL WARFARE LABORATORIES
FORT DETRICK, FREDERICK, MARYLAND

1 April 12,?9
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((II) Y1Wrlew fen!t) Trinidad #5917 straiiiZ~!.U'.,:J: : .
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:! 1,(U) SOURCE: Trinidad Regional Virus Laboratory, Port of Spain, Trinidad.
-(._.:..,.} - . 5"00/~
!'•,(U) HISTORY: ,1Virus was originally isolated trom a human lethal case of
yellow fever that occurred in Trinidad during thet},954 outbreak. It was received
on 23 February 1955 as a frozen preparation of i-i.f.tf parcent human serum suspended
in bovine albumin. This preparation was subsequently employed to produce suf-
ficient infectious material '.(by infection of Rhesus monkeys) for research and
laboratory infection of mosquito vectors.

_... IT/
~(U)~ The etiologic agent of yellow fever is considered one of the
small animal~viruses. By the use of collodion membranes of graded porosity
•,"(grado~l membrane~). j.ts diameter has been estimated to be from 17 to 25 milli-
microns •. (1) Casals·C2) has classified the yellow fever virus as a Group B arthro-
pod-borne virus on the basis of its antigenic properties in hemagglutination
reactions. It persists in nature intracellularly within a number of animal hosts
and mosquito vectors in endemic areas of the world. The virus may be cultivated
J:!! .!!!2 in several animal hosts but may undergo modification in virulence, tissue
affinity, and/or pathogenicity by passage in some animals, particularly mice.
Cultivation in monkeys does not appear to modify the virus to any significant
extent but non-discernable changes might result from repeated monkey passage al-
though such changes have not been demonstrated. Prolonged cultivation of yellow
fever virus in tissue culture may result in marked reduction in virulence for
humans and monkeys.
( f,,) ,-( )
1
• ,(U) b·. The virus of yellow fever is extremely labile.' 1 It is
readily inactivated by heat, usual antiseptics, carbon dioxide gas, O. 9 peNCBL cJI ~
sodiUDf. chloride, Loek.e's solution, Ringer's solution, hormone broth or distilled
water.,(1.3,4,5) With the exception of the antiseptics, deleterious actions of
these media on the virus are reduced if 10 p~ieat, o1.more, blood serum or 0.75)'~
,-:aaae bovine plasma albumin is added to the diluent.,(3) The viability of the
virus is maintained for long periods of time when stored in sealed, glass ampules
and kept at -10°c.',(4) The best method for preservation of the virus is 4~si-
ccation while in the frozen state and storage in a refrigerator at - 70°c.,(l)
r~1 I
.e::
In the Rocky Mountain Laboratories of HEW, on a Fort Detrick
~'(U) .
contract, it was found that yellow fever virus ',(Trinidad #5917 strain) in ·wet
monkey plasma could be stored undiluted in sealed lass ampules with carbon _
i.,v,,f- t c) 7 'L- ~

1
Ft. Detrick Control
El/- .5- t(JJ (7{>) No.69.-FD&-.39?

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( ' A • 1' / ' -J._
 dioxide refrigeration at -70°C fpr at least 2 years without loss of titer or
virulence for monkeys or mice.'•(6J Approximately 1500 ml of plasma containing
yellow fever virus with a mean log titer of 9.3 mouse ICLD50 per ml was stored
at -7o 0 c in 30 ml quantities in sealed, glass ampules. At various time intervals
over a 2-year period, aliquots were removed and the amount of virus present
determined. No decrease of titer was observed in the test samples.

,.-(u) Master seed: One 1,(1) ml of

a.
intraperitoneally into a Rh~ monkey. On the
of plasma was obtained from the infected monkey
designated as RM#52 first monkey passage, had a
of virus per ml computed in accordance with the
to produce a working seed.
serum ffJ'JJ./ was .1.uu"'u ...a. .. c.u
fourth day of infection, 50 ml
by cardiac puncture. This plasma,
titer of 1 x 108 mouse ICLD50 •s
Reed-Muench method and was used

',(C) JI.' Working seed:

'.°(C} ~
Six to eight pound Rhesus monkeys were infected by
intraperitoneal inoculation of 1 ml of undiluted RM#52 plasma. Inoculated
monkeys were held in non-ventilated monkey cages with the open wire mesh cage
fronts continua.lly subjected to UV-irradiation. When sympJ:i)ms of infection
\(fever and general appearance) became pronounced ~ t o ~ days after inocu-
lation, the monkeys were anesthetized by intraperitoneal inoculation of nembutal
•,(sterile solution NEMBUTAL sodium. 50 mg per ml. Pentobarbital sodium. Abbott.)
and exsanguinated by cardiac puncture using sterile technique. Reparin ':(Hynson,
Wescott, and Dunning, Inc.) was used as an anti-coagulant. Approximately 10 ml
of a solution of one~(l) gram Heparin per 500 ml sterile physiological saline was
added to each 500 ml centrifuge bottle prior to adding monkey blood. Heparin
solution was filled into syringes and needles to be used in exsanguinating monkeys
and then expelled from the~ syringe leaving the needle full.
(.t..L
1.-(c) c;f;JIJ'. The heparinized blood was centrifuged in the cold
f(5°C) at 20,000 r.p.m. for 15 minutes in an International Refrigerated Centrifuge,
Model PR-2. Plasma was drawn off from the packed erythrocytes, dispensed in 3 ml
aliquots in pyrex ampules which were then sealed and frozen in an alcohol-dry
ice bath and stored at -700 C in a dry CO2 chest. The amount of virus present was
determined by titration of aliquots of plasma by intracerebral inoculation of
21-day old Swiss mice, Detrick strain. The results of the titrations and amount
of those monkey plasmas subsequently used in preparing a working stock are shown
in Table 1. Approximately 100 ml of plasma can be obtained from each monkey.

2
 rqgjwt1110R. ,..--Adult female mosquito, Aedes 1,(Stegomyia) aegypti ',(Linnaeus), j
Detrick strain. ' /
j
~, '."(U) DESCRIPTION:
- . (J.I
,(U) ~ The adult female Aedes aegypti is a small-to-medium sized
1

species of mosquito ranging in body length from 3 to 6 mm. It is marked with

silvery-white or yellowish-white bands and stripes on a nearly black background.
It has a "lyre-like" pattern dorsally on its thorax, i.e., two outer curved
yellowish-white lines and two medium parallel lines. The legs are conspicuously
banded, and the last joint of the hind leg is entirely white. The head is covered
with broad flat scales with only a single row of upri§ht forked scales. The
species is widely distributed within the limits of 40 N and 40°s latitude. In
the u.s. it occurs as far north as Norfolk, Virginia, but does not survive the
winters north of this latitude.

'.'(U)1..--1
The Aades aagypti mosquito is a domesticated species, '
breeding in close pro~im.ity to, if not actually inside, human habitations. The J
eggs are deposited singly, usually in containers at or near the water line. The
adult tends to stay close to its breeding focus and most remain within 200 m
of their point of origin. However, the Aedes aegypti mosquito appears to seek '
.j

'
out human habitations and more extended flight ranges have been observed when
adults were released some distance from. occupied communities.
. [,J
i
i

\{U) c. Unlike the adult male, the female adult mosquito takes 'i
periodic blood meals from. animals; thus the capacity to transmit yellow fever is
limited to the female. The female adult~- aegypti preferentially bites human
I
!
beings, takes a blood meal two days after emergence and approximately every three
days thereafter. ---while probing. for . a blood meal the infected female mosquito
transmits~ow fever to a suitable animal host. It has been concluded on the
basis of \(a) increase of virus titer in the mosquito, and •,'(b) requirment for an
extrinsic incubation period for yellow fever transmission by.roosquitoes infected
as adults that transmission of the disease is a biological process rather than
a mechanical one.

'.(U) fJ!
The specific vector proposed for infection with yellow fever
virus is that resulting from the colony reared and maintained at Fort Detrick
in accordance with the protocol outlined herein.

3
 1,(U) y.II Gener 11 l: The production sequence parallels the life history
of the mosquito. Fertilized female mosquitoes lay eggs which hatch into larvae.
Development then proceeds through four larval instar stages and a pupal stage.
Adult mosquito/a emer~e from pupae to complete the life cycle.
(,_
/
 (U) LITERATURE CITED~
l. Theiler, Max. "Yellow Fever") in Viral and Rickettsial Infections of
Man, Rivers, T. M. and Horsfall, F. L., Editors. 3rd Edition. J.B.
Lippincott Co., Philadelphia, Pa., pp. 343-360. 1959.
2. Casals, Jordi. "Viruses: The Versatile Parasites. I. The Arthropod-
Borne Group of Animal Viruses. 11 Transactions of the N.Y. Academy of
Sciences. 10:3:219-235. January, 1957.

3. Bauer, J. H. and Mahaffy, A. F.: "Susceptibility of African Monkeys to

Yellow Fever." Amer. Jour. of Hyg., 12:155-174, 1930.

Theiler, Max: "Yellow Fever, The Virus, 11 in Yellow Fever, George K. Strode,
Ed., 2nd Edition, McGraw-Hill Book Company, Inc., New York, pp. 50-54, 1951.

5. Sawyer, W. A. , Lloyd, W. D. M. , and Kitchen, S. F. : "Preservation of

Yellow Fever Virus." Jour. Exptl. Med., 5<1(1):1-13, 1929.

~~ Technical Study 7, Part B. Short Title: XYA-8121. Entomology Division,

Fort Detrick, Md. July 1958. SECRET'.' 58-FDS-887).
 MAY 18 l'db~
TO: Secretary, Chemical Corps Technical Committee, Building #1,
Army Chemical Center, Maryland

1. (U) Subject document is forwarded your office for appropri-

ate action.

2. (U) At the present time, no specification for ei~her the

agent or the agent-vector combination has been prepared. However,
depending upon the availability of sufficient data for specification
and surveillance criteria, it is estimate4 that these documents can
be completed within six to nine months. Three documents \ld.11 be in-
volved: (1) a specification for the agent, (2) a specificatiQn for
the agent-vector combination, and (3) surveillance criteria for both.
 ( U ~ The early yellow fever research carried out by the Yellow Fever Com.-
mission(5} and others< 0 ,7) demonstrated that laboratory-reared and laboratory-
infected Aedes aegypti mosquitoes are capable of infecting humans. Infections
have occurred when human volunteer subjects were subjected to the bites of from
one to fifteen laboratory reared mosquitoes which had previously fed on patients
clinically ill with yellow fever. That laboratory-reared and-infected Aedee
aegypti mosquitoes are capable of transmitting yellow fever to humans is further
supported by laboratory accidents reported in medical literature.(4}

2
 (U) .Q..• Yellow fever epidemics have been limited primarily to Africa and
the Americas although significant epidemics have occurred in western Europe.< 4)
Non-immune humans appear to be universally and uniformily susceptible to the
virus of yellow fever with the possible exception of Negros and the newborn.(12)
There is no evidence that Asians possess any racial resistance to yellow fever
and migrant 4\,sians have contracted the disease while working in Africa and the
Americas.< 14)

(U) c. Yellow fever has never occurred in certain areas of the world, for
example Asia, in spite of the fact that ecologically and meteorologically many
such areas appear favourable for the disease. It has been postulated that the
epidemiology of the disease does not permit transoceanic voyage from endemic
areas.(15) With the development of modern transportation facilities, public
health authorities have become increasingly concerned with the possibility of
introducing yellow fever into virgin areas. The demonstration of :immunological
overlap between arthropod-borne viruses of the same antigenic group has sug-
gested an alte~native hypothesis.(16) It might be possible that a population
becomes partially resistant to yellow fever as a result of coumon infections
by one or more related viruses. Dengue is known to be prevalent in some areas
of the world, particularly southeastern Asia. Furthermore, it has been shown
that Dengue virus is capable of eliciting an immune response in experimental
an~ls that is somewhat protective against infection by yellow fever virus.(l6)
itioual information is required to determine whether a human population that
been exposed to infection by a related virus(es) will show resistance against
asion by the virus of yellow fever. At the present time, neither theory is
ved nor disproved.
 -
(U) h.• The efficacy of yellow fever vaccination with the l7D strain in
preventing infection of humans subjected to mosquito bites is indicated by
the fact that subsequent to the introduction of prophylactic vaccination in
the Americas and Africa the incidence of yellow fever has been significantly
reduced.(20,21) With few exceptions, cases reported in areas where mass-
vaccination was properly practiced have been timited to the small portion of
the population which had not been vaccinated.< 22 ) Most of the cases reported--
in vaccinated humans have been explainable on basis of the period of time~
required for protection to develop (5-12 days) subsequent to vacciTJ.~.t..ion.-
/\,,
 (U(~} The storage stability of uninfected kedaa EM. psi,-mosquitoes is
influenced by many factors; including density of mosquitoes per unit volume,
density of mosquitoes per unit area, temperature, relative humidity, number of
air changes, type and frequency of food supplied, type material and configeration
of container, availability of blood meals during storage, and genetic stability
of the particular strain of mosquitoes being stored.(11)