SPECIAL VIRUS - LEUKEJIA PROGRAM

PROGRESS REPORT #h

PAGE

OVERALL SUIMARY 1

SEGMENT CHAIRMEH'S COMBINED SUMHABIES . .

I Developmental Research ITII Testing and Monitoring 22III Resources and Logistics 2hIV Epidemiology 29V Special Animal Leukemia Ecology Studies 33VI Biohazards Control and Containment 38

APPENDIX

List of SVLP Contractors iiX

SPECIAL VIRUS-LEUKEMIA PROGRAM

OVERALL SUMMARY

F. RA.U5CHER AND R. RETSINGER

The probability is very high that viruses responsible for at leastsome humaa sad domestic animal leukemias will be found, and thatit will "be possible to develop measures (including test vaccines)for the prevention or control of these diseases. These predictionscould not have been made several years ago and are based largely onrecent findings through work conducted within the Special Vims-Leukemia Program (SVLP) of the National Cancer Institute.

Beginning in 196 , the Congress of the United States provided fundsto the National Cancer Institute (NCI) for an intensified programin virus-leukemia research. These appropriations have "been madeto support specie! efforts and to complement the extensive researchfunded through the usual mechanism of grants in support of work byother investigators.

Convergence Technique

was formulated. The funding level foi

>ociai;ea witn avisn ana rourine j-eujtemia are e^iO-Logicaj, agentsof human leukemia, and (2) to develop an effective vaccine or othermeans for the prevention and/or control of humsn leukemia andlymphoma when such etiological agents are found- The main assumptionor working hypothesis on which the overall Program is based is thatat least one virus is an indispensable element for the induction{directly or indirectly) of at least one kind of human leukemia(including lymphoma) and that the virus and/or virus genome persistsin the diseased individual.

From the research standpoint the program is divided into 1* majorareas of effort: Human Leukemia Etiology and Prevention, SpecialAnimal Leukemia Ecology Studies, Biohazards Control and Containment,and Human Leukemia Therapy. Approximately 100 of 18U projects,which currently make up the program plan, are being conducted by

investigators in governmental laboratories and clinical facilities,in universities, in nonprofit laboratories, and in commercialfacilities. Selection, implementation end continuation of allprojects are done on the basis of both scientific excellence endhigh priority relevance in terms of the integrated program.

B. Scientific Activities - Summary Fy '67

The Special Virus-Leukemia Program was planned and implemented in1961+ and was based on the justifiable conviction that sufficientscientific facts and leads, and, technical competence were availableto mount a special, intensive, coordinated program -- a programspecifically aimed at the prevention and effective treatment ofhuman leukemia and lymphoms. The validity of this conviction andthe definitive scientific base of the program have been significantlystrengthened through inhouse (NIH) and contract research developmentin Fy '6?-

Biometry. In addition, data and guidance from the laboratorieDrs..Robert Huebner, NIAID, Alan Rabson, LCP, John Fahey, LI,Paul Gerber, DBS, and Seymour Perry, Edward Henderson, and PaulCarbons, MB have contributed very significantly to the program.

General. -- Contract projects within this program are chosen andimplemented on the basis of technical and intellectual excellence,need, and relevance to specific program objectives. While, therefore,the measurement of virtually all reported data and information areof immediate or predictable relevance, their individual citationand analysis are at this time premature and in this report unnecessary.It is, however, entirely pertinent to discuss the modification andelimination of old end the development of new avenues of approach.

A Scoreboard of biologic agents most frequently detected or isolatedfrom human leukemia and lymphoma patients to 19&7 "would list myco-plasma, unidentified Herpes-type virus, "C" type virus (morphologicallyidentical to avian and murine leukemia viruses), cytomegsloviruses(also Herpes-type) and Reo viruses (particularly type III).

One avenue of approach thst has been significantly curtailed concernsstudies on mycoplasma as etiologic candidates for human leukemia endlymphoma. In 1966 and through early 19 7 > investigators of 8

- 3 -

different contract laboratories were heavily committed to mycoplasmastudies. While in some cases this appeared to be warranted, 8considerable portion of their efforts were being diverted away fromvirological aspects of the program. The decision to abandonfundamental studies on mycoplesma was based on (a) no spparentdifferences between the amount and kind of mycoplasma associatedwith cancer patients and patients with other non-neoplastic diseaseSj(b) the demonstration that mycoplssma-were often present in laboratorypassaged specimens as adventitious or inadvertent contaminantsderived from aerosols from laboratory animals and people snd fromtrypsin and serums used for tissue cultures, (c) the demonstrationthat murine leukemia and Rous sarcoma viruses known to be free ofPPLO will following inoculation into SPF or germfree mice, alsoknown to be free of PPLO, induce typical progressive leukemia orsarcomas, and (d) the demonstration that virus free of PPLO isrecoverable from these induced diseases. Because of these findingsand because of their apparent role as ubiquitous contaminants, itwas decided to limit work on mycoplastna to the monitoring of leukemicmaterials which are to be used for virus isolation attempts.

Persistent infections with cytoraegaloviruses have been reported in arelatively high percentage of leukemic children and to a lesser extentin leukemic adults. These viruses ere usually detected in urine buton few, if any occasion, are they recovered from neoplastic cellsor tissue. These viruses heve also been detected in a relativelylarge number of children with other diseases (e.g., infectiousmononucleosis) and, of course, in children with cytomegalic Inclusiondisease.

Reo viruses (particularly type 3) have long been recognized ashaving an extremely wide spread host range (man, mammals, fish, birds,etc.)- British investigators have detected Reo type 3 virus in upto 10% of African Burkitt lymphoma patients. Preliminary studiesby Dr. Stanley, Australia, suggest the possibility that Reo virusisolated from a Burkitt lymphoma patient may be capable of inducinglymphomas in a very small percentage of laboratory mice. To ourknowledge these reports on the detection of Reo virus in lymphomapatients and the possible induction of lymphoma in a mouse havenot been confirmed. Reo virus has not been detected in lymphomamaterials derived from African patients and worked up in the UnitedStates. Similarly, the virus has not been detected in tissue culturesestablished from approximately TO American patients with leukemiaor lymphoma. Efforts will, however, be continued within the SVLPto detect and, if warranted, characterize Reo viruses from cancerpatients.

The virus or group of viruses most persistently detected and isolatedfrom human leukemia and lymphoma materials is s Herpes-type agent.Morphologically, the particle is identical to known members of theHerpes group including Herpes Simplex, Herpes Zoster, end cytomegaloviruses. This virus(es) appears to be an important candidate forconsideralion as en etiologic agent of human cancer for the following

reasons:

1. Largely within the past two years we haveestablished tissue cultures of cells from the buffy costsand tissues of over 60 patients with malignant disease(predominantly leukemia or lymphoma). Approximately li-2of these lines are known to be infected with a Herpes-type virus. The absence of virus from the remaining linescannot be definitely established without more exhaustivestudy because the maturation of the agent is usuallyobserved in a small fraction of the cell population. Theselines have been established by 25 investigators in variouslaboratories and from patients of 5 continents. Thedifferent virus isolates appear morphologically identicaland at least in some cases appear to share common antigens.

2. This herpes-type virus(es) has not been foundto be related to any of the known herpes viruses of animalsincluding man.

3- In addition to its presence in established tissuecultures, HTV has also been found prior to tissue culturein "buffy coat and brain cells of human leukemics, and inat least 1 case, also in peripheral leukocytes of themother of 3 congenital leukemic.

k. Several of these cell lines have already beengrown in industrial quantity wherein it is possible toproduce kilogram quantities for subsequent virus extraction.The virus can be recovered, purified and concentrated tc*the extent that preparations containing approximately ICr" to10 particles per nil are available for study.

5- Herpes virus(es) of identical morphology isassociated (perhaps etiologically) with adenocarcinomas offrogs and with lymphomas of the African clawed toad. Acell line started from the; peripheral blood of a Rhesus monkeywith acute myeloid leukemia also contained a herpes-typevirus. More recently, tissue cultures were started fromeach of two young juvenile chimpanzees following theirinoculation with two of the human cell lines known to containHTV. Both of these chimp lines were heavily infected witha HTV. These findings in chimpanzees may represent (a) thedetection of an indigenous chimp virus having morphologicaland other characteristics in common with human HTV, (b) thetransmission of human HTV (for the first time) to an animalrecipient, or, less likely (c) survival of HTV containinghuman cells in peripheral chimp blood.

6. A specific neo-8Htigen (G) has been found repeatedlyto be associated with many human tumors and human cell linesof malignant origin. With only one exception this antigen

had not been found in cells of nonmalignant human tissuesor in nonmalignant cell lines. A recent study showed thatG-a.ntigen was induced in normal human amnion cells by herpesvirus A (a recent human isolate) but not by vaccinia virus.Whether or not a cause and effect relationship exists betweenherpes infection, appearance of G-antigen, and consequent orsubsequent malignancy is not known. In this regard, severalother recent studies appear pertinent. The induction ofmurine papillomata following inoculation of herpes-virus Aend 3-methocholanthrene is directly dependent on the amountof infectious virus contained in the synergistic inocula.The survival time of human diploid cells heterotransplantedto the hamster cheek pouch is increased after infectionof the cells with several viruses. Six patients who developedrecurrent herpetic lesions around the mouth also developedsubsequent carcinoma at the sites of the herpetic lesions.If G-antigen is in fact a specific factor related to tumors,these results further strengthen the hypothesis that certainherpes viruses may be oncogenic.

T- The original report by Fink, et al., confirmed byinvestigators at the University of Michigan and at RoswellPark Memorial Institute, showed that conjugated antiseraprepared against pellets of human leukemic plasma known tocontain C-type particles could be used to detect and monitorthe presence of an immunofluorescent antigen in the buffycoat and bone marrow cells of about 60-70$ of patients withleukemia. This immunofluorescent reagent also reacts withthe same percentage of cultured cells which by electronmicroscopy is shown to contain the herpes-type virus. Noreaction (fluorescence) is observed with cells infected withherpes simplex, cytomegalo and other human or animal herpesviruses.

The program area in which we have experienced greatest difficulty isthat concerned with the detection and replication of C-type particlesfrom human leukemia and lymphoma. Experiences with animal neoplasmsindicate that there are at leas 22 different viruses etiologicsllyassociated with the induction o leukemia and lymphoraa in murine,avian, feline, snd probably in anine and bovine species. Thisand other information suggests hat if some human leukemia andlymphornas are virus, induced, th most likely etiologic agent willbe of the C-type. During 1967 we continued to show that the plasmasand in some cases tissues of approximately 30$ of human patientscontain C-type particles. These particles, however, apparentlydo not survive or persist in an identifiable form in tissue culture.Conversely, herpes type virus is found seldom, if at all, in plasmabut is very frequently found in tissue cultures derived from biopsytissue and from peripheral leukocytes of cancer patients. Whileit may be somewhat difficult to understand how virus or virus-like

particles which differ substantially in morphology, site of maturationa d presumably in chemical composition caj be etiologically relatedto the same group of diseeses (leukemias and lyraphomas), a similarphenomenon occurs i. animals. Fibrosarcomas of nearly identicalgross a .d microscopic morphology can be induced in hamsters withboth polyoma (MA) and Rous sarcoma (RNA) viruses. It is, of course,entirely possible that the herpes-type virus is an internationalubiquitous passenger virus with no etiologic relationship to neoplasia.Until this is determined, however, a large proportion of the resourcesand effort of the program must be devoted to studies with this virus.The obligation to follow the herpes lead does not mean that effortswith C-type particles will be curtailed. The elusive C-type particleis still considered the prime candidate, and newer leads emergingfrom model systems will be expeditiously exploited and applied tostudies of the human disease.

Major scientific activities which have had to be conducted andaccomplished for the prevention and/or control of microbiologicallyinduced diseases are presented in Table 1. These sequential activitiesappear to be as applicable to the control of human leukemia andlymphoma as they were to the vaccine prevention of smallpox, whoopingcough, polio and measles, etc.

Table 1 presents information on the principal model systems availablefor virological studies on leukemia or lymphoma and attempts to reflect^according to these sequential activities, the current statue of effortstowards the control of these diseases in animals as well as in man.Since the discovery by Sllerraann a^d Bang in 1909, that a chickenleukemia was transmissible by an "ultravisible virus" fifty-sevenyears have elapsed, during which time 3 viruses (or principal groupsof viruses) have "been found to "be associated with avian leukosis.To date no effective method for the prevention or control of leukemiain chickens is available. However during the 15 years since Gross'discovery of the cell-free transmissibility of murine leukemia,approximately 16 viruses have been isolated and several effectiveways have been shown for the prevention end control of murine leukemia.Forms of this disease in raurine animals can be prevented effectivelyby vaccination and the disease can be controlled by genetic manipulationof the host to some extent by chemotherapy and by intercepting variousforms of horizontal transmission (containment of infected or carrieranimals, prevention of feces ingestion, cannibalism, and ingestionof infected milk). A model system which may allow significant studiesrelevant to the findings of herpes-like viruses in human leukemiaand lymphoma cell lines is that reported by Balls, et al_. concerningthe apparent association of a herpes-like virus with "spontaneouslymphomas" of the African clawed toad (Xenopus species). This animalis common in the Burkitt lymphoma aree of Africa and as reported byHaddow, et_ al., mosquitoes which bite man in that area also, onoccasion, feed on toads.

MEASLES

POLIO

1930

H I I-

55T"1—>ls>

1965

1965

1951

1909

8 U

I I3 3

' g

In man the beginning of a major effort to detect and characterizeviruses from human leuKeraic materials began in 1957 with Dmochowski'sfindings of "C" type particles in the lymph nodes of human patients,and, in 1963 following reports by Epstein, e_t sj . and Grace, et si.of herpes-like viruses in cell lines of Burkitt lymphoma and chronicmyeloid leukemia patients, respectively. If one assumes that (a) oneor both of these virus types are etiologically related to the diseasein man, and that (b) reports of studies conducted with these candidateviruses are valid then the overall progress towards sequential stepcontrol is, for type "C" particles -- between the steps of isolationand replication; and for the herpes-like particles -- "between thesteps of characterization and industrial replication.

Table 1 also presents information which compares progress towardsthe prevention or control of leukemia in animals and man, to non-neoplsstic human diseases for which effective vaccines have recentlybeen developed and used- Approximately 58 years elapsed between thedemonstration that polio was a virus-induced disease and the developmentof an effective vaccine for 3 entigenic types of virus. Similarly,it took approximately 3 years to develop an effective vaccine,with 1 virus type, for measles. It is to be noted that similarongoing studies with "C" type and herpes-like virus particles ofhuman leukemia have been conducted for 9 and 3 years respectively.

Progress Highlights. -- Several of the most significant facts andleads from studies conducted within or related to this programpredominantly during Fiscal Year 196? are as follows:

1. PROGRAM FLAMIHG AMD MANAGEMENT -- A very high effectivelevel has been attained in the coordination of like scientificactivities conducted by investigators in Government, universityend commercial laboratories and clinics -- and in the iden-tification and integration of different activities relevantto the objective of the total program. Specific decisioncriteria for each major scientific area have been identifiedwhich must be met prior to systematic advancement from onescientific phase into another.

This is imp_grta_nt_Jbgcaus^ they allow, within aprogram motivated in part "by a strong sense oftime urgency and for which funds, personnel, and.space are not unlimited, clear identification of(a) projects of highest excellence, need andrelevance, (b) less fruitful avenues of approach,(c) areas of desirable (and undesirable) dupli-cation, and (d) projects which are scientificallysound but which even if performed successfullywould not aieasurably contribute to satisfyingdecision criteria and therefore, attainment ofoverall program .oh }ecbives.

DETECTION AND ISOLATION OF VIRUSES — It is now apparentthat virus-lilte particles of two different types can bedetected, and in some cases isolated, from patients ofdifferent countries who are afflicted with ell kinds ofhuman leukemia or lymphoma. Particles of one kind ("C"type) are identical with those known to cause leukemia inlaboratory animals. Viruses of the other type are similarin size and shape but not identical to those known tocause fever sores or shingles in man, and lyraphomas andcarcinomss in toads and frogs.

This is important because the repeated detectionand isolation from patients of viruses similarto those known to cause cancer in anima-ls is --in itself -- indirect suggestive evidence ofvirus involvement in human cancer, and is pre-requisite to the growth of larger quantities ofvirus, in the laboratory, for studies which maylead to a vaccine.

GROWTH OF HUMAM GANGER CELLS IgJTHE LABORATORY -- SVLP con-tractors and others have firmly established the growth oftumor cells in the laboratory from 105 different patientswith leukemia, lymphoraa and other cancers (See Table 2).Eighty of these tissue culture cell lines were derived fromchildren and adults of Africa, Australia, England, Sweden an<the United States suffering with leukemia or lymphoma. Theremaining 25 tissue cultures were begun from living tumormaterials obtained from American patients with other formsof cancer.

This is important because we can now grow thosequantities of living human cancer cells in thelaboratory which are necessary for virus isolationattempts. Since these cancer cells reproduce innumber for many years, newer techniques of virusdetection and isolation can be applied to astandardized source of human material. It isno longer necessary, therefore, to rely com-pletely on the procurement of fresh patientmaterial for each laboratory experiment. Thisis of paramount importance.

RECOVERY OP VIRUS FROM_HIMAH LEUKEMIA AMD LYMFHOMA TISSUECULTURES -- Virus particles~of identical size and structurehave been seen in at least 60 of these human tissue cul-tures (See Table 2). We can now grow industrial quantitiesof these tumor cells and recover large quantities ofpurified and concentrated virus.

TABLE 2 -- SUMMARY: HTV AW HUMAN CELL "LIBES"

PATIENTSCATEGORY PATIENTS

CULTUREDATTEMPTED *

PATIENTS HTV -*• **• PATIENTS CULTURED

LINES HTV + **LINES CULTURED

Lyraphoma

Lyraphoid Leukemia

Myeloid Leukemia

Sarcoma -Carcinoma

TOTAL (CANCER)

Other diseases

Normal

TOTAL (NON CANCER)

No.

31/61

15/111

34/138

25/54

105/364

5/54

14/81

19/135

Percent

14*

25*

46*

29*

9*

IT*

14*

Ho.

19/31

4/15

T/25

44/105

1/5

3/14

4/19

Percent

61%

2T*

28*

42*

20*

21*

21*

No.

4/17

23/100

11/46

61/204

1/5

4/20

5/25

Percent

24*

23*

24*

30*

20*

20$

20*

HTV = Herpes type virus; (prototype = Epstein's EBl).* Approximate. Successful attempts understandably are nearly always

reported; nonsuccesses, usually not.** These numerators are not identical tecause (a) several positive lines

have "been started from the same patient and (t>) up to 6 lines have"been started" from the same patient of which only 1 or 2 arepositive.

This is important because it provides largequantities of virus from human patient materialswhich are needed to determine ability to inducecancer in animals, and comparisons of similaritiesand differences to other human viruses and toanimal viruses known to induce cancer in animals.

5 DISTRIEUTIOH OF_CANDIpATEjyiRUSE5 "IH NATURE" -- Herpes-typeviruses which appear to occur most frequently in leukemiaand lymphoma patients have also been found in horses, frogs,toads, snakes, monkeys, chimpanzees, and in people with non-cancerous diseases as well as in "normal" persons. Viralantibodies hsve been found in 10-80$ (depending on age) ofnormal people as well as in nearly 100$ of persons withcertain kinds of cancer. Virus particles have been foundin the blood and tissues of very young leukeraic childrenas well as of their parents, brothers and sisters. Herpes-type virus was recently found in blood cells of a childborn with leukemia gnd in blood cells of the normal (non-leukemic) mother.

This is important because it (a) indicates theubiquitous widespread distribution of this virusin nature, (b) suggests that e "normal" nondiseasedcarrier may "be a source of infection and diseasefor highly susceptible individuals, (c) suggestssimilarities to other human diseases such aspolio wherein approximately 95$ of people wereinfected although less than .0001$ developedparalytic disease, and (d) suggests that virusesassociated with human leukemics may be transmitted,before birth, from mother to offspring as areXncwn leukemia viruses of mice and chickens.An understanding of these phenomena is of utmostimportance to the feasibility and use of pre-ventative vaccines.

6- DEVELOPMENT OF SYSTEMS TO TEST LARGE NUMBER? OF HUMAflSERUMS -- Reliable and sensitive laboratory methods havebeen developed which will allow us to determine thenumber snd type of people in selected populations whohave been exposed to and possibly infected with theseviruses.

This is importan since it is not possible toobtain direct proof of the ability of a humanvirus to induce cancer in man — i.e., we cannot inoculate man deliberately with a suspecttumor virus. We can, however, obtain indirect

evidence or proof of cancer-inducing capability,by determining whether certain cancer patientsare infected with the suspect virus in highernumbers, or more severely than normal peopleor patients with other diseases.

RECOVERY OF VIRUS DIRECTLY FROM PATIENTS -- Collaborativestudies conducted between laboratory and clinical investi-gators have made it possible to collect very largefractions of blood suspected of containing leukemiaviruses. This is done by separating and retaining theplasma and "by returning the essential blood cells to thepatient. This has mede it possible to detect and recoverlarger quantities of virus directly from differentpatients than previously were available.

This is important because it will allow compar-ative studies"of~~the "C"~~type particles (whichto date can not be grown in the laboratory)recovered from leukemic human plasma with theherpes-like viruses derived from the 60 differentcultures of human leukemia or lymphoma cells grownin the laboratory. It will also allow comparativestudies of human "C" type particles with similarviruses known to induce leukemia in laboratoryand domestic animals.

ATTEMPT TO TRANSMIT HUMAN LEUKEMIA TO LABORATORY ANIMALS --It has now been established that at least one tumor virusknown to cause cancer in chickens and other bird specieswill induce malignant sarcomas in marmosets, a species ofsmall monkeys. Materials from human patients have alreadybeen inoculated into 600 newborn monkeys and chimpanzeesof various species, and into large numbers of dogs, cats,mice, hamsters, etc.

This is important^ because the leukemia or tumor-inducing ability of human viruses must bedetermined in a non-human animal -- preferablyone which is phylogenetieally close to man.Successful induction of leukemia in animalswith a specific human virus will also pro-vide a test system for more effective utili-zation of drugs and evaluation of proceduresfor the treatment of human leukemia.

EXPANSION OF STUDIES ON AFRICAN AMD AMERICAN LYMPHOMAS --It ""is now firmly established that while Burkitt lymphomaoccurs in African children at ac incidence higher than acombination of all other forms of childhood leukemia andlymphoma in Africa, the disease also occurs in a smallbut significant number of individuals in the United Statesand other countries. In Africa this disease occurs inchildren who live predominately in geographical areascharacterized by certain temperature, rainfall, andaltitude limits. Because of the incidence of thisparticular tumor, and because some of the characteristicsof the disease strongly suggest that an insect vector may"be involved in the transmission of the Africen disease,•we have established and are coordinating close workingliaisons with key scientists and clinicians in Africaand in the U. S. In order to continue these workingrelationships and to more effectively feed back infor-mation to our African colleagues, a member of ourprofessional staff will spend a year in Africa for thepurpose of lecturing to medical students, of obtainingclinical specimens for studies in laboratories of theNCI, and to set up a controlled epidemiologlcal study.

This is important because it establishes a firmcollaborative study with scientists andclinicians knowledgeable about a most unusualchildhood lymphoma which currently is consid-ered most likely to be associated with acausative virus. The establishment of anepidemiological survey is of utmost importancebecause certain regions of the Burkitt lyraphomaarea in Africa may be used as one of the fieldtrials for testing a candidate vaccine.

SOME TUMOR VIRUSES REQUIRE HELPER VIRUSES -- A highly potentmouse sarcoma virus was discovered this year. The mejorsignificance of this new laboratory model is the findingthat this virus requires the help of a leukemia virus (dualinfection) for the Induction of malignant sarcomas. Thesestudies parallel those previously reported with severalsarcoma viruses of chickens and suggest that the helperphenomenon may contribute to the occurrence of tumors inother animal species -- including man. To test thishypothesis tissue cultures have already been establishedfrom 5 human sarcomas, the microscopic type of which isidentical to tumors induced In mice vith the Moloney sarcoma

virus.

This is important because it may open the way fordisclosing causative viruses associated withhuman sarcomas and may provide a new avenue ofapproach to the search for human leukemiaviruses.

PRODUCTION OF EFFECTIVE VACCINES FOR MOUSE LEUKEMIA --It is now recognized that at lea'st 16 virus'strains' arecapable of causing leukemia in mice and rats. It appearsjhowever, that similar to polio in man, these viruses fallinto only several different antigenic types. It hasalready been shown that a vaccine prepared against oneparticular virus will immunize mice not only against thatvirus but against others as well. As has recently beenaccomplished with polio and measles viruses, both liveand killed vaccines have been prepared with mouse leukemiaviruses. In some cases the vaccines and/or their anti-bodies together with drugs are reasonably effective whenadministered, to leukemic animals.

This is important because it shows that (a) animalleukemia can in fact, be prevented with vaccinesand that the causative viruses from which thevaccines were prepared can be attenuated and/orkilled by procedures already established for theinfluenza, polio, and measles diseases of man,and (b) it is possible to treat (by vaccine orcombination immuno-chemotherapy) mice afterleukemia develops. This approach may makepossible more effective control of the humandisease than that attained by drug therapy alone.

IMPLEMENTATION! OF COLLABORATIVE CONTRACTS TO STUDY LEUKEMIAIM CATTLE. GATS AND DOGS -- New and unique facilitieshave been constructed and are currently being used todetermine the number and type of leukemias and lymphomasin ca.ttle, cats and dogs which are caused by viruses andto determine whether the newborn of these species, aswell as monkeys, can be used as sensitive indicator hostsfor candidate leukemia viruses recovered from man. Wehave already succeeded in establishing lines of leukemiacells from cattle and dogs which can be transplanted intothese animals with the causation of leukemia. Electronmicroscopic studies of the transplantable tumor of cattleand of an established tissue culture of a dog lymphomahave revealed the presence of large numbers of type "C"virus particles identical in size and shape to thoseviruses known to induce leukemia in mice and rats.

These findings are important because theystrongly indicate that many of the leukemiasand lymphoraas which occur in animals thsthave a prolonged and very close associationwith man throughout his life are also virus-induced. If this is indeed proven to be true,then means can be implemented for the preven-tion and/or control of animal leukeraias andpossibly of some human leukemias- It will bepossible to isolate, treat or sacrificeanimals known to be carriers of their ownleukemia viruses and to improve, if necessary,,currently used methods of treating consumableproducts, such as milk, milk products, andmeat.

13- INSECTS AMD ANIMAL TUMOR VIRUSES -- It is known that mitesare capable of transmitting chicken leukemia viruses, andthat certain mosquitoes can transmit avian sarcoma virusesfrom one chicken to another. Species of kissing bugs,bedbugs and cockroaches are capable of maintaining infectioumouse leukemia virus for periods up to 72 hours. Mostrecently tumors were induced in fruit flies with the Roussarcoma virus of chickens. Studies are being conductedto determine whether these cancer viruses actually grow(replicate) in insects or whether the virus simply survivesbetween blood mea-ls and is therefore transmitted throughthe mechanical process of biting-

These studies are important because they mayprovide (a) clues on how tumor viruses aretransmitted in nature, (b) added credence tothe hypothesis that Burkitt lyraphoma ofAfrican children is insect transmitted, and(c) a means for controlling the incidenceof some cancers similar to the control ofmalaria through eradication of mosquitoes.

1 . BIOHAZARDS AMD CONTAINMENT -- During the past year it wasshown that highly potent laboratory strains of mouseleukemia viruses can be carried in the air and thatinhalation, by mice, of contaminated sir results in infec-tion and leukemia. To protect animals, laboratory workers,and other people from possible disease by viruses known orsuspected of causing leukemia, air filters and systemshave been developed with a 99-99$ efficiency.

This is Important because it alerts us to theneed for protection of laboratory -workers andanimals from potential infection with uncommonlystrong preparations of viruses studied in thelaboratory. The concurrent finding that lowdoses of virus (roughly equivalent to thosefound in nature) were not transmitted throughair is also import a trt_ because it providesadditional indirect evidence that humanleukemia probably is not contagious.

17

DATE- February 28S 1967

SPECIAL VIBUS LEUKEMIA PROGRAM

Segment Chairman's Combined Summary

Triannual (Annual) Progress Reports

Period Covered: _p_ct_ob_er_ 1, 1966 through January 31_, 1967

Program Segment: Developmental Research

Included in Combined Summary:

Getmfree Life Research Center (PH43-65-95) - Germfree Life andOntogenesis.

Chas. Pfizer & Co., Inc. (PH43-66-98) - Development of VirusTumor Test Systems.

Children's Hospital of Philadelphia (PK43-66-477) - Interferenceand Immunofluoreecence Studies with Cell Lines Derived from Leukemiaand Lymphona.

Gerrafree Productss Inc. (PH43-64-533) - Production of MurineLeukemia Seed Stocks.

Health Research, Inc. (PH43-63-593) - Electron Microscopy forStudies of Viruses in Human Leukemic Cells.

H. D. Anderson Hospital and Tumor Institute (FH43-65-604) -Studies on the relationship of Viruses to Murine Leukemia andLyrnphoraa,

Health Pesearch, Inc. (PP.43--65-616) - Development of a Large Volumeof Leukemic Cells.

University of Texas Dental Branch (PH43-65-628) - Hunan Leukemia-Lymphoms Study.

The Regents of the University of Michigan (PE43-65-639) - ContinueResearch Relating to Etiology of Human Leukenia.

Blonetics Research Laboratories, Inc. (PH43-67-661) - Investigationof the Carcinogenic Activity of Selected Virus Preparations inNewborn Monkeys.

18

11. Microbiological Associates, Inc. (PH43-67-697) - Evaluation ofHuman Tumors in Newborn Mice and Bioassay of Animal Tumor Virus.

12. Rutgers University (PH43-65-1039) - Conduct immuno-chemical andantigenic studies of known and candidate leukemia viruses.

Segment Chairman:Robert A. Manaker, Ph.D.

Segment Vice Chairman:

Kurine leukemia virus studies continue at a reduced level. Productionof the Rauscher and boloney strains from mouse plasma at the John L. SmithMemorial for Cancer Research; Chas. Pfizer and Co., Inc., remains unchanged.However tissue culture mouse virus production was discontinued with 12liters of 1'oloney virus and 2 liters of Rauscher virus in storage. Theprogram at Germ Free Products. Inc. to produce mouse leukemia viruses inSPF mice for immunological investigations provided adequate amounts of FriendRauscher and Koloney viruses. 30 percent of requirements for Ereyer-Holoney.Hanaker and Buffet viruses, and some rat passaged Pauscher and Boloneyviruses.

Quantitation of Pauscher strain virus by application of the latexparticle method showed that virus concentrations of 2-5 X 10 virus particlesper ml applied to F.M. screen grids in accordance with routine negativestaining procedures resulted in an average distribution of one particle pergrid square. This provides a basis for ready estimation of virus contentof samples. Virus assay has also been continued in mice to quantitate leuke-mogenic activity. During the past quarter. 75 bioassays were completed and60 new assays were initiated.

Immunological studies showed the Rauscher and Moloney strains of viruschemically coupled to sheep red blood cells as a carrier were highly immuno-genic in mice and rabbits. The viruses were readily inactivated. Commonantigenic determinants among the Rauscher, Moloney, Friend and Gross virusstrains were demonstrated by immunodiffusion. Ether treatment of theRauscher virus strain yielded RNAase-ser.sitive nucleoids infective for tissuecultured cells and newborn mice but not reactive in neutralization or com-plement fixation tests.

A normal mouse bone marrow cell line, designated V9 and maintained inlong-term culture, was shown to be susceptible to infection by the Rauscher,Friend, Moloney and Gross virus strains. Uninfected V9 cells had undergonea change to malignancy demonstrated by the formation of localized tumorsfollowing infection into mice. When V9 cells were infected with Sauschervirus., their ability to grow as a localized tumor was lost. The phenom-enon is under study.

The V9 culture inoculated with Rauscher virus-infected mouse tissueand designated V10 has been used for virus production. As previouslyobserved with virus produced by the V5 culture, virus yielded by cultureV10 lost leukemogenic activity. The change in this property was rapid atabout the 60th serial subculture of the cell line. The V10 virus is notas effective as is V5 virus in inducing mouse resistance to subsequentchallenge with leukemogenic Rauscher plasma virus and may, therefore, differfrom it in some respects.

The effort to detect, isolate and characterize viruses which may be ofetiological significance in human leukemia has expanded during the lastquarter. Six new cell lines developed at Chas. Pfizer & Co., Inc., from

I-U

fifty-four blood or other tissue specimens, all carried a herpes-type virusmorphologically similar to that found in cultured Burkitt lymphoma cells.At Health P.esearchs Inc., ninety-five new white blood cell lines wereestablished from fifty-five donors. About one third contained the herpes-type virus. Sixteen of the lines were started from eleven non-leukemicdonors. Three of these lines carry the herpes-type virus.

At the University of Michigan, activity is directed to the search forC-type particles using four established human cell lines as host culturesfor virus. Electron microscope monitoring of inoculated cultures disclosedtype C particles in a few cultures which could not be verified as virus inthe absence of observation of budding. Bone marrow specimens from 228leukesiic and fifty-three non-leukemic children as well as eighty-six leukemicand fifty-six non-leukemic adults were tested in tissue cultures. Someviruses were isolated and identified as adeno-, herpes, or parainfluenzaviruses. One isolate is as yet unidentified. Direct examination by electronmicroscopy of bone marrows or sedimented plasma specimens detected no virus.Direct cultivation of bo«s marrow, lymph node, and white blood cells frompatients with and without leukemia and subsequent exposure of the fourestablished human cell lines to these cells was conducted. Unequivocalcytopathogenic changes were observed in sone cultures so exposed to cellsfrom leukemic donors. Kycoplesma could not be implicated. Direct cul-tivation of 181 specimens of tissue from patients with leukemia was alsocarried out at the M, D. Anderson Hospital and Tumor Institute. Fifteencultures remained viable after 100 days of incubation. Two of four bonenarrow cultures cultivated for two months contained particles resemblingthe C-type. Direct electron microscope examination disclosed particlesresembling C-type in six lymph node specimens from six patients. Thissuccess may be ascribed to the exhaustive search conducted in this lab-oratory .

The production of herpes-type virus from a culture of the AfricanBurkitt lyraphocia has been increased at Chas. Pfizer and Co., Inc. . by asubstantial increase in the virus content of the culture. Other Pfizercell lines reportedly produce comparatively high yields of herpes-typeviruses and promise to be useful for comparative studies. Techniquespracticed at Health Research, Inc., involving modification of culturalconditions have also effectively raised virus yield in the Africanlysphoma culture.

The mixed hemaglutination test is being applied at M. D. AndersonHospital and Tumor Institute to sera from patients with leukemia in theexpectation that some will contain antibodies against candidate virusesor tumor antigen. These sera would provide tumor specific reagents forfuture studies.

Immunofluorescence (FA) tests conducted at the Children's Hospitalof Philadelphia using selected human sera showed that positive fluorescenceof cultured cells from patients with Burkitt lynphoma, leukemia and some

normal individuals was accompanied by intracellular herpes-type virusdetectable by electron microscopy, strongly suggesting that the fluorescencewas specific for viral antigen. The herpes-type particles in the P3JC'Jiyoye' ) and EBB cell lines could not be identified with herpes simplex,cytomegalo or varicella virus. Indirect FA tests against P3J and EB3 cellswith sera frcm twenty chimpanzees, forty-six baboons, nine rhesus monkeys,ten normal and lyraphomateus cattle, seventy-one normal dogs and thirty-one lymphonatous dogs were negative. Weakly positive FA tests were obtainedwith sera from four primates that had received injections of Burkitt lymph-oma materials. The Fiak/Malmgren FA technique was used at the Universityof Michigan to check 100 bone marrow specimens. Marrow cells from eightypercent of patients with acute lymphatic leukemia and thirty-three percentof patients in remission were positive while marrows from control patientswere negative. A strongly positive marrow was obtained from a patient withprostatic carcinoma.

Human sera from different donors and suitably absorbed rabbit antiserato the herpes-type particles associated with lymphotna cultures were usedat Chas. Pfizer and Co.„ Inc. to demonstrate coating of naked herpes-typevirus particles from different cell lines. Coating of herpes simplex virusby these sera was not observed. The sera from cancer patients were morestrongly reactive than those from normal individuals.

The primate testing program provided 133 live births with only elevenpercent mortality during the reporting period. At peEsent 879 breedinganimals of nine species are on hand1 755 animals sre in holding. Ninety-five newborn animals were inoculated.

Examination of oral and anal swabs of newborn primates for agentscytopathogenic for monkey kidney culture yielded seventy-one adenoviruseSjthirty-six entercviruses,, and three unidentified agents. A colony-widesurvey using immunoflucrescenee methods is underway to specificallyidentify Vibrio species isolated. Vibrio^ fetus may be responsible forabcrtions and infant deaths.

Studies initiated include the inoculation of two immature chimipanzees,one with the AL-2 cultured American lymphoma cells and extracted virus andone with F132 cells and virus. The latter culture was originated frcm apatient with reticulum cell sarcoma. Cultures started from the peripheralblood of these animals three days after inoculation of the viable human cellscontained cells morphologically similar to those inoculated. The herpes-type virus was observed in these cells insuring that opportunity to infectthe primate host with the agent was maximal.

In another study,, nineteen newborn monkeys were inoculated with freshly-Irawn whole blood from patients at the' NIH Clinical Center. A specialiroject in progress involves induction of malaria in the recipient prior•n innrnl/it-Lon with Burkitt virus.

22 May 15, 1967

SPECIAL VIRUS-LEUKEMIA PROGRAM

SEGMENT CHAIRMAN'S COMBINED SUMMARYof

TRIANNUAL (ANNUAL) PROGRESS REPORTS

Period Covered: October 1 , 1966 - January 31 , 1967

Program Segment: TESTING AND MONITORING

Contracts Included in Combined Summary:

1. Baltimore Biological Laboratories (PH43-62 -839)Development of Fluorescent Antibody Reagentsfor Selected Mycoplasma Strains

2, Baltimore Biological Laboratories (PH43-63-1161)Fluorescent Antibody Studies of Mouse Viruses

3. Baylor University (PH43-63- 590) -- Studies on Virusesas Related to Cancer with Emphasis on Leukemia,and Continuation of the Testing Program in Primates

4. Chicago Park District (PK43-65- 101 7) -- Marmoset BreedingCo lony

5. Flow Laboratories, Inc. (PH.43-65-634) -- VirusLaboratory for Cancer Research

6. Wistar Institute (PH43-65- 102 8) -- Human Leukemia-VirusTest System: Problem Survey

7. K'istar Institute (PH43-65- 1002) -- Study of Role ofMycoplasma Isolated from Human Leukemia Specimens

Segment Chairman77

Segment Vice-Chairman v^'

A comprehensive viral diagnostic laboratory is being established atFlow Laboratories under the direction of Dr. James Duff. Reagents arebeing prepared for identification of viruses associated with diseases ofman and other animals. It is planned that appropriate reagents will beadded to the laboratories' armamentarium to increase and broaden itsscope and capabilities. — Antiserums prepared in other laboratories againstthe "C" type and herpes-type viruses found in material from human leukemia/lymphoma patients have been tested at this facility for neutralization andCF reactivity against several recognized human and other animal virusesincluding members of the enterovirus, myxovirus and herpesvirus groups.No significantly positive results have been obtained to date.

An imciunofluorescent ^plaque neutralization technique for lymphocyticchoriomeningitis virus has been developed by Dr. H. Wilsnack at the BaltimoreBiological Laboratory.

A project to begin in July, 1967 is designed to test the reliabilityof different serological techniques in differentiating between leukemic andnormal cells, or between cells containing the Burkitt-associated virus, andthose not containing the virus. The most reliable technique will be selectedfor a proposed sero-epidemiologic survey of human populations. Dr. Benyesh-Helnick, of the Baylor University contract, has developed one of the tech-niques to be tested.

SPECIAL VIRUS-LEUKEMIA PROGRAM

SEGMENT CHAIRMAN'S COMBINED SUMHAPYof

TRIANHUAL (ANNUAL) PROGRESS REPORTS

Period Covered: October 1, 1966 - January 31, 1967

Program Segment: RESOURCES AND LOGISTICS

Contracts Included in Combined Summary:

1. California, University of (PH43-63-13)Mammali an Cell Culture Laboratory

2. Connecticut, University of (PH43-62-505)Establishment of a Specific Pathogen Free Flock ofWhite Leghorn Chickens

3. Flow Laboratories, Inc. (PH43-65-1012)Low Temperature Specimen Repository

4. Hazleton Laboratories, Inc. (PK43-65-635)Rauscher Leukemia Virus Production

5. Hospital For Sick Children (PH43-6S-97)Procurement of Plasma from Acute Leukeiric Patients

6. Hyland Laboratories (PH43-66-894)Facility for the Procurement and Distribution ofSterile, Unfiltered Fetal Calf Serum

7. Hyland Laboratories (PH43-66-539)Serum Testing Laboratory

8. Louisville, University of (PH43-66-902)Preparation of Simian Foamy Virus Reagents andAntisera

9. Fakerere University College (PL43-67-47)Epiderciologic Study of Burkitt's Lynphoma

10. Massachusetts General Hospital (PH43-65-1034)Preservation of Platelets and Leukocytes by Freezing

11. i'elpar, Inc. CPK43-65-641)Human Embryonic Tissue and Cell Biology Facility

12. Melpar, Inc. (PH43-65-1033)Moloney Virus Production

13. Montreal Children's Hospital, The (PK43-65-1020) m-2Procurement of Plasraa fron; Acute Leukemic Patients"

14. Notre Dame, University of (PH43-65-1018)Development of Leukemia Virus-Free Gerrnfree MouseColony

15. Southwest Foundation for Research and Education(PH43-66-950)

The Production of Simian Virus and Homo logous Antiserun1

16. Tulane University (PH43-64-881)Evaluation of Tree Shrew as Tumor-Virus PesearchAninal

17. University Laboratories, Inc. [PH43-66-1133)Rous Sarcoma Virus Production

Segment Chai

Two contracts, Hospital for Sick Children, Toronto, anciMontreal Cfii Idrens Hospital, which provide clinical specinensfron untreated acute leukemia cases were requested to supplyserums rather than the previously supplied plasma. Outputfrom these sources will form part of the s erum collectionnecessary for a comparative study of new immunologi cal tests.

Contributing to the serum collection will be the MakerereUniversity contract which is providing sera from Burkittlymphona patients, their family, and control sera. Acombined epidemiological and descriptive study of Burkittlymphoma in Uganda, this contract will serve as a nucleus fora coordinated program to study that lymphona as a dises.seproblem and not simply a source of material useful to thelaboratory. In FY-6S for ex amp le, the NCI Che me therapy Areawill send a chemotherapy team to Uganda tc uncover, describe,study and treat tiie disease as it occurs in that are?-.

ContleukstilprogHospandlasttagareprotfor

ing sttes inepende

is b1 centteletsr aretechn

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A contract with Melpar fur a Human Embryonic Tissue and CellBiology Facility is also oriented toward the cryobiclexicalfield in the processing cf human perinatal kidney and lungtissues which are used extensively in virus isolation andas say pn cedures by contractors and col lab or at in n; scientistswithin the SVLP and virus cancer programs of HCI, Frozen,characterized and concentrated suspensions of embryonic eelIsensure c. continuing and dependable source of material f '> rpertinent pr'jprnrss.

A i'ielpar contract as well as contracts with Hazelt^nLaboratories and Has on lie search Institute suppl ied 1 arptiquantities of Rauscher and Molc.ney nurine laukenia virusconcentrates to the SVLP. A product, extensively monitoredfor extraneous viruses, is available thrcuph the utilizationrf the VCB virus testinf service. The mice used in virusproduction are also routinely tested.

Ili-fc

The pr;blen of latent viruses in 1abc ratory animals continuestc stimulate further research and development directed towardobtaining virus-free and virus-defined penetic stccks. TheUniversity of Connecticut contract has provided a source c. fchickens and epps free from mest poultry pathogens. Theproblem of Marek's disease has proven very difficult but hopecf a possible solution has been raised by the generation cf 2small nucleus stock so far found tc be free rf the disease.The eggs in excess cf breedinp needs fron this project havebeen suuplied not only tc virus cancer investigators but alsotc the Division cf Biologies Standards for use in neaslesvaccine as s ays.

At the Lobund Laboratory, University of Notre Dame, furtherattempts have been made to find strains ._ f mice free frcmlatent murine leukemia viruses. All .domestic strains cflaboratory mice examined tc date have shown evidence cfleukemia-like virus in their thymic tissues. Viruses havenet been detected in a strain cf wild mice and a strain oflaboratory nice, XIII, from France. Work en these willcontinue since the presence of latent leukemia virusesincreases the difficulty in interpretation :>f the resultsof every experiment us inn mice ?.s hosts for human or aninalleukemia experiments.

Pxublens of latent aqents and extraneous microbialcontamination are not confined t~ laboratory animals butalso include the cell cultures derived from aninr.ls or nan.

The Cell Culture Collection Committee ein 1960 has effectively dealt with thesinception and new continues under spcnsType Culture Ccllecticn tc add new charto its repository. The University of CBirlcrical Laboratory ccntract has beenprc^ram by supplying nen-primate mammalscientific community. Developnent cf ncultures was success fully undertaken fcAnimal Leukemia Ecclufy Studies wcrkinrcell lines from denestic and wiId animup cur armamentarium of reagents with wvirology.

This ccntract has also provided research support tr anotherResources anti Lc^istics pronran, that cf defining and impr^.vthe widely-used fetal calf serum.

stablished by the NCIproblems since its

rrship of the Americanacterized cell linesalifornir. - Naval

cornerstone of thisian cell lines tc- theeeded be,vine cellr the SVLP Specialsegment sind otherIs continue to builuhich to- clc C'. aparative

Stu.y ,festablissif*nif icseven seantibodyfetal claboratrevealedparticleelectronparticlequestionti cell

in .ivi-.ualod that S3nt amountsruns ca p a i n s t b (j

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ni crcscupyremains t

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alf seruDS conterns .

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cal Labn s c . n tt w L ^ fecifice, testin am.tricll'sirus-liinet. bythese

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These finCinrs support the belief that ccntinusl refinementand characterizatiin cf bitlt-vjically Derivev' materials useiin research is a practical necessity. In _rcer tv ..;; this,two contracts v/ith Hylan^. L?.b(. ratcries [PH43-66-539) ani"(PH43-66-894) prcviceO fi:r ;;rn.ucticn an^' tcstin"capabilities fcr fetal calf serum. The ains cf this -jr*.'-ranhave been to Reveler;- criteria ant", specifications fcr anaccent able ana reliable se run useful f<,r bcth cell culturean>- virolexical studies. Since a tissue culture virL-lu'icallaboratory will use from 50-100 liters i,r cicre cf thismaterial per year it is im^crtant that increas in'-ly stringentquality standards be eve; Ivei".

Final ly, contracts f •.. r ^-rc t'ucti: n i.f sinian virus reagentshave pr->viJe,". aJ.i_iticnal tec Is f L r ,'etecti: n an... i.'.cntif icr.ti _ni,f this irapcrtant TLUP cf viruses. Sinian virus rea~untprc ..ucticn shr.ui;. be cL-rap 1 etoJ. by 1969 an/ will became a bnsicrescurco fir primate investi/ati.ns.

IV-1

DATE: March I, 196?

SPECIAL VIRUS-LEUKEMIA PROGRAM

Segment Chairman's Summaryof

TRIAHNUflL (ANNUM) PROGRESS REPORT

Period Covered: October Jt, 1966,- January 3.1j J9&7

Program Segment: Epidemiology

Contractor: National Center for Health Statistics (D.C.)

Contract Number: FS-(66)-35

Title: Deaths Certificates on Childhood Cancer, 1960-1964

Contractor's Project DIrector: Dr. Robert D. Grove

Project Officer (NCt): Robert W. Miller, M. D.

Objectives: To obtain death certificates for all U. S. children under 15 years

of age who have died of cancer, 1960-1964. This registry wil l be

used to determine 1) aggregation of cancers In twins or other

sibs, 2) geographic clustering by dates of birth or death, 3)

the association of cancer with congenital defects, and k) the

frequencies of specific cancers not separately listed In pub-

lished mortality statistics; and 5) the registry wil l also be

used to match against names of children believed to be at un-

usual risk of cancer; e.g., those given certain vaccines In the

past.

Date Contract Initiated: Fy-1967, "

Current Annual Level: $8,500

Total Funding Committed to Date: $8,500

Progress: Work completed. About 22,000 copies of death certificates have been

received.

Problems: None

Projections: Copies of corresponding birth certificates wi l l be sought from

each State. The data from the birth and death certificates wil l

be abstracted and card-punched; lists and tables wi l l be made

to obtain the objectives described above.

The file wil l be up-dated at two-year Intervals by obtaining

childhood death certif icates since the previous cut-off date.

IV-3DATE: March 1, 196?

SPECIAL VIRUS-LEUKEMIA PROGRAM

Segment Chairman's Summaryof

TRIANNUAL (ANNUAL) PROGRESS REPORT

Period Covered: October I, 1966 - January 31. 1967

Program Segment: Epidemiology

Contractor: National Academy of Sciences-National Research Council

Contract Number: 43-6^-¥», Task Order No. 23

Title: Investigations of Viral and Other Factors In Cancer Etiology In ArmyVeterans

Contractor's Project D!rector: Mr. Seymour JabIon

Project Officer (NCI): Robert W. Miller, M. D.

Objectives: 1) To study the cancer experience of 23,1^9 soldiers following

World War II In relation to diagnoses made during military

service and suspected to increase or decrease cancer occurrence;

e.g., infectious mononucleosis, Infectious hepatitis and herpetic

Infections.

2) To study about 3,200 veterans who developed cancer, 1950-195**,,

to determine If frequencies of certain events during military

service, 19^2-1944, exceed those for a matched control group;

e. g., blood transfusion and yeJlow-fever vaccination.

Date Contract Initiated: June 1, 1965

Current Annual Level: $34,900

Total Funding Committed to Date: $65,900

Progress; Excellent. Progress reports reveal that study is on schedule and

going as planned.

Problems: Kcno.

Projections: Should finish on time.

SPECIAL VIRUS-LEUKEMIA PROGRAM

SSGMEM? CHAIRMAN'S COMBINED SUMMARYof

TRIANKUAL (AICTUAL) PROGRESS REPORTS

Period. Covered: October 1, 1966 - January 31, 1967

Program Segment: SPECIAL ANIMAL LEUKEMIA ECOLOGY STUDIES

Contracts Included in Combined Summary:

1. University of California (PHS 3-65-609) - - Bovine LeukemiaTransmission Studies.

2. Cornell University (Pi&3-65-620) - - Research on Susceptibilityof Cat to Viral Leukemogenesis.

3. Michigan State University (PHU3-65-100) - - Human/Canine LeukemicTransmission Trials: Etiology of Canine Leukemia.

it. University of Pennsylvania (H&3-65-1013) - - Research inExperimental and Natural Transmission of Bovine Leukemia.

5- Research Foundation of the State University of Nev York(H&3-65-605) - - Comparative Studies on Structure of KnownViruses and Articles Observed in Bovine Tissues.

6. Einstein Medical College, New York (PHU3~65-6l2) - - Researchon Immunological Factors in Susceptibility to Murine leukemiaViruses.

7- Taft Sanitary Engineering Center, Cincinnati, Ohio (JSS-VCL-30)Studies on Thermal Inactivation of Viruses in ML1& and Milk Products.

8. Institute for Medical Research, Camden, Nev Jersey (PS43-65-65)To Conduct and Etiological Study on Bovine Leukemia.

9. University of Minnesota (FHU3-65-606 - - Research on Susceptibilityof Cows to Known Tumor Viruses.

10. University of North Dakota (PHU3-66-8) - - Insects as PossibleExperimental and Natural Vectors of Murine and Avian Tumor Viruses.

Segment Chairman

Segment Vice-Chairman " A, (. / .

3U v-2

Research efforts conducted within the Special Animal Leukemia Ecology

Segment of the Special Virus-Leukemia Program are concerned vith determining

the etiology of leukemia as it occurs in certain domestic animals. In addition,

a primary effort of this Segment is to determine the relationship, if any, of

animal leukemias to lymphoma and/or leukemia in roan. Studies within the SALES

program may be grouped as follows:

RE5EARCH_ig_BOVIjSE LEUKEMIA. Dr. Robert Marshak, University of Itennsylvania,

is maintaining an "experimental" bovine lymphosarcoma by whole-cell transplant

of the neoplasm in irradiated calves. The role of known carcinogenes in the

induction of bovine leukemia is being studied. It is hoped that these materials

will serve as virus as virus source tissues. Reciprocal foster nursing experiments

have begun to clarify the relationship "between ingestion of "viremic" milk and

the occurance of bovine leukemia or lymphocytosis. This researcher has established

in tissue culture five cell lines derived from leuekemic cows. These lines are

in stationary and/or spinner cultures. Three lines are of buffy coat origin;

two lines are of lymph node origin. The cells are lymphocytic in appearance and

aneuploid. The lines are to be checked for the presence of virus particles by

electron microscopy.

Dr. Ray Dutcher, Institute for Medical Research, Camden, New Jersey

is collaborating with Dr. Marshak in the latter study. In addition, this

researcher has established and partially characterised a continuous kidney line

from a leukemic cow. It is believed that this line will contribute to standard-

ization of virus interference studies and quantitation of interferon-like activity.

Dr. Dutcher is conducting electron microscopic surveys of bovine mammary gland

tissue. Virus particles have been found in a certain number of these specimens.

The particles were usually found in ducts and/or vesicles vithin the cell.

Dr. Charles Burger, Upstate Medical Center, State University of New York,

is also collaborating with this group. He has accomplished separation of Type C

particles from the milk of leukemic cows. These have been isolated in large

35 V-3

enough quantities to permit definitive characterization of the particles. These

virus fractions have been extracted for nucleic acid and the data indicated that

RNA is present which is presumably of the single-stranded nature. This researcher

is also collaborating with Dr. George Moore's group, Roswell Bark. He has examined

large volumes of tissue culture fluid from Eurkitt cell lines. Herpes-type

particles have been recovered in relatively large numbers. Chemical data

on these fractions have been obtained indicating a preponderance of DMA.

Dr. Gordon Theilen, University of California, has been able to establish

a good correlation between onset, degree and persistence of lymphocytesis in cows

with concentration of inoculated normal and/or leukemic bovine buffy coat materials.

Although none of the animals inoculated with tumor tissues have developed leukemia,

many have extreme blood dyscrasis. Electron microscopy of pellets derived from

leukemic plasma of inoculated animals have revealed particles, some of which

resemble the Type C particle. Cytogenetic studies conducted by this investigator

on animals which received tumor whole-cell material indicate a chromosomal

structural change related to persistant lymphocytosis. In some animals the

number of cells with polyploidy was increased above the normal.

Dr. D. K. Sorenson, University of Minnesota, has inoculated 26 calves vltb

selected nmrine leukemia viruses. Response of the animals to the viruses is being

followed through hematological, electron microscopic, and seriological studies-

Intrauterine fetal inoculation of the leukemia viruses is also being studied in

k& cows bred by artificial insemination. Both the Moloney Leukemia Virus and

Rauscher Leukemia Virus appear to infect a continuous line of bovine endocardia!

cells as determined by direct fluorescent antibody techniques.

Dr. R. E. Read, Taft Sanitary Engineering Center, Cinncinati, Ohio, has

developed thermal destruction rate curves for the following viruses: Herpes-simplex

l Adeno-12, Sv- 0, Reo 1, Rauscher and Moloney Leukemia Viruses. A tissue sample

from a human leukemia patient maintained "in vitro11 has yielded plaque forming

agents. This material will be studied by the National Cancer Institute staff for

viral characterization. Engineering studies are progressing which will result

in the development of more effective pasteurization procedures. During the

next year, virus inactivation in sewage will be studied in collaboration with the

Biohasards Segment of the Special Virus-Le'ukemia Program,

RESEARCH IH GANIN3 LEUKEMIA. The principal effort in this area is concen-

trated at the Michigan State University, Lansing, Michigan under the direction of

Dr. Gabel Conner. Here twenty types of candidate leukemogenic materials, both

human and animal derived, have been inoculated into 226 Caesarean-derived neo-

natal Beagle dogs housed in isolation quarters. Eight percent of the dogs which

have received leukemic whole-cell or cell-free dog material have developed malignan"

tumors. Some dogs, including those which received human inoeula developed a

lymphadenopathy. Eetero-species induction of malignant lymphosarcoma (reticulum

cell sarcoma) occurred in one of eleven animals which received Moloney sarcoma

virus. Electron microscopic studies conducted by these investigators have describe

intracytoplasmic crystalline arrays of particles, l8 to 22 mu in diameter in

malignant tissue of 100$ of the induced and 90$ of the spontaneous cases of canine

lymphoma. Antigenic studies using immunofluorescence have shown that human

gamma globulin reacts positively with Burkitt cells. Serologic studies on dogs

with lymphosarcoma have shown the presence of specific complement fixing antibody

which reacts to human mumps antigen. The host cell-virus infectivity spectrum

has been determined for canine thymus cells using certain candidate viruses.

RESEARCH _IH FELIME LEUKEMIA. Dr. Charles Rickard, Cornell University

has been able to transmit lymphosarcoma using cell-free preparations from an

American cat to three nev-bom animals of the same species, Type C virus particle;

have been found budding from the plasma membranes of one cat with spontaneous

lymphocytic leukemia. They appear morphologically different from the known cat

viruses. A tissue culture cell-line has been established from a cat neurofibro-

sarcoma.

3T V-5

IHSECT_y_ECTOR STUDIES. Dr. Robert Fischer, University of florth Dakota,

has shown that selected blood-sucking insects can transmit the Twiehaus virus

from infected chickens to normal hosts. The virus is viable vithin the insect

for at least 72 hours. The blood-sucking insect, Triatoma infestans as well as

Bedbugs,can transmit Rauscher Leukemia Virus from viremic mice to normal hosts

of the same species. This virus remains active in the infected insects for an

extended period of time. Studies will be extended to include the Moloney Sarcoma

Virus because of the rapid bio-assay methods now available for this agent.

jjENITIC S7UDIES_. Studies on genetic and immunological factors which play

a role in the susceptibility of animals to tumor viruses are being conducted

by Dr. Frank Lilly, Einstein Medical College. In the Friend virus leukemia system

there are indications that the H-2 type influences the ability of animals to

respond immunologically to the virus antigens. Experiments to study levels of

antibody production in relation to E-2 type are now in progress. In both the

Friend and Gross leukemia virus systems other genes effect susceptibility or

resistance. The differentiation according to host strain specificity of two

strains of Friend Virus has been completed.

Program

38

SPECIAL VIBUS- LEUKEMIA PROGRAM

Segment Chairman's Combined Summaryof

TBiamUAL PROGRESS REPORTS

Period Covered: October 1, 1966 - January 31, 1967

'nt: BIOHAZARDS COHTROL AND COHTAIHMEHT

VI-1

DATE; March 1, 196T

Contracts Included in Combined Sui lary:

1. Hie Dow Chemical Company (PHlf3-65-10 5)Research and Development on Biohazards Control and containmentFacilities

2. U.S. Department of the Army, Ft. Detriclt (WCI - FS - (66) - 31)Evaluation of Filters iri-th Sub-Micron Aerosols

3. The Ohio State University Research Foundation (PHl(3-65-lCOl)Hazards of Experimental LeuKemia Research.

k. University or Minnesota (P1A3-65-999)Development of an "Open Isolation" System for the Care ofLow Resistance Patients.

:nt Chairman

Sequent Vice-Chairman

Under the research and development contract on biohazards control and con-

tainment, several significant areas have been investigated,

Automatic V7atering Devices: Of the three commercially available valves tested

to date, only one valve prevented passage of Pseudomonas^ aeruginoga to un-

inocul'eted mice for periods up to seven days. More extensive tests are

planned.

Mobile Virus_Laboratory: Construction of the trailer and laboratory compon-

ents is on schedule and will be completed during remainder of FY 19 7 •

Simulated^Laboratory and Safety Cabinet.Evaluations: Smoke evaluation has

been completed and preliminary studies of particle size distribution of

Serratia marcescens from various virus laboratory accidents have been completed.

Prototype Laboi- torles: Preliminary designs for a virus concentration lab-

oratory and safety monitoring laboratory have been submitted to HCI for review

and approval.

Biohazards Warning Symbol: Decals, use-stsndards and posters are currently

being evaluated by NCI laboratories and selected contract facilities. The

symbol has been adopted by the Agricultural Research Service, USDA.

The studies which are being conducted with ultrahigh-efficiency filters (HKPA)

challenged with T^_ bacteriophage (0.1 micron HMD) will provide additional

information concerning the correct, safe application of these filters in

laminar flow safety cabinets and rooms for virus research and patient core.

In addition, studies are in progress to determine aerosol transmission in

four viral-host systems, i.e., 1) newborn hamsters and type 12 adenovirus

2) BALB/C mice and Rauscher virus, 3) yearling monkeys and Yaba virus and

k) newborn marmosets or monkeys and Rous-sarcoma virus. Results of these

studies thus far indicate that natural transmission of oncogenic viruses,

particularly Rauscher murine leukemia virus, by aerosols is a definite

possibility.

The evaluation of the concept of horizontal laminar flo\* as a pstient

isolation system has shown that the concept is feasible. Additional efforts

will be expended to define design criteria and operational procedures for

such ti facility.

SVLP COFTRACTOBS

Athey., ".- EALB/c v'ouse Colony, fMicrobiological Associates, Inc.,Walkersville Maryland., FHfc3--66-911*; Program Hanap;e»ent.

Bernstein, E. Rous Sarcoma Virus Production. University Labora-tories., Highland Park, New Jersey PHl*3- 66 1133, Resources andLogistics Program Se*$ment.

Biddle, J. L.• Preparation of Feline Virus Reagents and Antisera.Dow Chemical Company (Pitman--Moore Division)., Zionsville; Indiana,PHU3 67 657 Resources and Logistics Program Serpent.

Burger C. L.: Comparative Studies on Structure of Known TumorViruses and Particles Observed in Bovine Tissues. Research Foundationof State University of I'ew York, Syracuse, ITew York. PHU3-65 -605 .SALES Program Segment.

Carski, T. R.- Development of Fluorescent Antibody Tests for J-iycoplasma. Baltimore Biological Lab... Inc.. Baltimore, Maryland,PHH3 -62 839 , Testing and Monitorinp Program Segment.

Cascardo., M. • Private Viral Diagnostic and Reference Reagents Lab. •Human Leukemia Virus Lab. Flow Laboratories, Pockville , Maryland,PH**3 67 1135•, Testing and Monitoring Program Segment.

Conner., G. ' Human/Canine Leukenic Transmission Trials: Etiology ofCanine Leukemia. Michigan State University, East Lansinp, Michigan,PHU3-65-100 SALES Program Segment.

Darte, J. M,, Human Leukemia and Normal Tissue Collection and Preservation. Hospital for Pick Children, Toronto.. Canada, PHt(3-65-97;Resources and Logistics Program Segment.

Decker; H. ".• Study of Filters with Submicron Aerosols. Departmentof the Anny, Ft. Detrick. Frederick , Maryland, FS--37-J BiohazardsControl and Containment Propraia Segment.

DeTendi, V,: Problem Survey of Hunan Leukemia Virus Test Systems.Wistar Institute, Philadelphia, Pennsylvania, PH^S-65-1028, Testingand I'onitorinp Prop-ram Segment.

Denton. P. L.' Supply of Leukeiviic and Normal Blood Plasim Specimens."Montreal Children's Hospital, Montreal, Canada, PH 3-65-1028K Testingand ?ionitoring Prop.rair Segment.

Doll F_. R.- Preparation of Antigens and Antisera for Equine HerpesViruses. University of Kentucky. Louisville, Kentucky, PHU3-67-llUO.Testing and Monitoring Program Segment.

13. Dno chows ki ., L, • £?••', Virological. Tissue Culture and Imnur-ofluores-cence Studies of Human Leukeciia/Lypiphoma Tissues ;*ycoplasmaMonitoring. University of Texas, Houston, Texas, PHl*3-65-iO"OU,Developmental Research Prograr' Segment.

', Dutcher R, • Etiological Studies of Bovine Leukemia,. South JerseyMedical Research Foundation, Catnden., New Jersey, PH 1*3-65-65. SALESProgram Segment.

15- Sdington, G. H.r Virological Studies of Burkitt Lymphons Specimens.University of Ibadan, Ibadan Nigeria PHlt3-67-6T^, Resources andLogistics Program Segment.

16. Farrow, W. • Production of i'furine Leukemia Virus Seed Stocks. GermfreeProducts. Inc., St. Petersburg, Florida, Ptib3--6k 533,, DevelopmentalResearch Program Seosnent.

17- Fischer, R. G, Insects as Vectors of Tumor Viruses. University ofEorth Dakota, Grand Forks North Dakota, PHU3-66--8, SALES ProgramSegment.

18. Fisher L. E, - Marmoset Breedinr Colony. Chicago Park District,Lincoln Park Zoo. Chicago Illinois, PHU3-65-1017 . Testing and "oni-toring Program Segment.

19. Gaeta. L. S.• Fetal Calf Serum Testing Laboratory. Hyland Labora-tories Los Angeles, California., FHl*3-66-539, Resources and LogisticsProgram Segment.

20. Oaeta. L. E.- Procurement of Fetal Calf Serum.. Hyland Laboratories,Los Angeles , California, PHU3 66-891*. Resources and Logistics ProgramSegment.

21. Gillespie, J. H.- Preparation of Feline Virus Reagents and Antisera.Cornell University. Ithaca New York, PHU3-67-66t, Resources andLogistics Program Segnent.

22. Gori G. B.- Boloney Leukemia Virus Production. Melpar^ Inc.. FallsChurch Virginiaa PHli3-65-lQ33, Resources and Loristics Progran Segment.

23. Gre.ce. J. T.: EH Studies of Viruses in Leukemic Tissues and Plasma.Health Research. Inc. . Buffalo, Eew York, PH113-63-593, Testing andTllonitoring Program Segment.

2k. Griesemer, R. A.- Aerosol Transmission of Oncopenic Viruses,OhioState University, Columbus, Ohio,, PHl*3-65 -1001, Biohazards Controland Containment Program Segment.

-.1*3-

25. Groupe, Vincent- Etiology of Marek's Disease. Rutgers, The StateUniversity. Eew Brunswick Hew Jersey, PKU3-67-11661 SALES ProgramSegment.

26. Grove3 R. D.: Death Certificates on Childhood. Cancer> 1960-1963.National Center for Health Statistics. PHS, Washington, D. C., FS-35,Epidemiology Program Segment.

27. Hayflick.; L. : Study of Role of Mycoplasma Isolated from Human Leu-kemia Specimens. Wiste.r Institute, Philadelphia.. Pennsylvania, PE*i3 •65-1002, Testing and Monitoring Proerram Segment.

28. Henle, G.: Interference and Immunofluorescence Studies of BurkittLymphoma Cell. Children's Hospital of Philadelphia. Philadelphia,Pennsylvania, PHU3--66--l*77; Developmental Research Program Segment.

29. Honicker, F. A.- Low Temperature Specinen Repository. Flow Labora-tories., Rockville, MarylandJ PH

it3-65--1012i Resources and LogisticsProgram Segment.

30. Euggins, C.~ Cyrogenic Preservation of Platelets and Leukocytes.Massachusetts General Hospital, Boston., Massachusetts, PHU3-65-103U,Resources and Logistics Program Segment.

31. Jablon, S.: Investigations of Viral and Other Factors in CancerEtiology in Army Veterans (Task Order Ko. 23). National Academy ofSciences, Washington, D. C., PKli3-6U kk, Epidemiology Program Segment.

32. Johnston. P. B. • Preparation of Simian Foamy Virus Reagents and Anti--sera. University of Louisville, Louisville, Kentucky, PHl»3-66-902,Resources and Logistics Program Segment.

3k. Levy B.? Evaluations of Human Leukemia/Lymphoma Specimens in Marmosets.University of Texas (Dental Branch), Houstons Texas. PHU3-65-628.Developmental Research Program Segment.

35. Lilly. F. - Research on IirirAiriological Factors in Susceptibility to.''urine Leukemia Viruses. Einstein Medical College. BronXi Rev York,PHU3 65-612, SALEP Program Segment.

36. Luginbuhl. R.' SPF Colony of White Leghorn Chickens. University ofConnecticut. Storrs, Connecticut. PHJ(3-62-505, Resources and LogisticsProgram Segment.

-111*..

Lutwana , J. S.s Collection of Burkitt African L;ir.phow.a Tissue Speci-mens, pakerere University Medical School, Uganda, PHif3-67-V7, Resourcesand Lopistics Program Se ent .

'"'adin 3.- Maraiali an Cell Culture Research. Propagation of T'orpalBovine Cell Lines. University of California. Oakland. California,PHU3 --63-13, Resources and Logistics Prop-ram Segment.

Madin, S. Facility for Univ. of California.. (63-13) Cell CultureResearch and Propagation. Naval Biological Laboratory., Oakland. Cali-fornia, FS-BS Resources and Logistics Proprar Segment.

i-'adison. R. Bioassays of Animal and Human Tissues, "'icrobiolopiealAssociates, Inc.., Falkersville. !7aryland_ PH^3- 6?- 697 . DevelopmentalResearch Program Se nent.

Marshak . R.- Establishment of Criteria for Normalcy vs. LeukemicAffection . University of Pennsylvania, Philadelphia, PennsylvaniaPHU3-65-629, SAL5S Program Segment.

Marshak R. Research on Experimental and Natural Transmission ofBovine Leukemia. University of Pennsylvania, Philadelphia, Pennsyl- ft*vania, PHi*3 -65-1013, SALES Prograc Segment.

i^attson . G. : Research and Develooraent re_ Biohazards Containment Facili-ties. Dow Cherlcal Co. . "idland/ Michigan. PKli3-65-10l*5 BiohazardsControl and Containment Prop-ram Segment.

Meier. K.: Studies of Natural Occurrence of vurine Leukenii- SarcomaComplex. The Jackson Laboratory, Bar Harbor, " aine. PH143-67-7**1* SALI:iS

Melnick. J. L.- Research on Viruses in Hurr.an and Primate LeukenicTissues and Plasma. Baylor University. I-Iouston, Texas, PHU3-65-590,Testing and Monitoring ProerairL Seraient.

Michaelson, G. S.- Develop Open Isolation System for Care of LowResistance Patients. University of ''innesota, "inneapolis .. Minnesota.PHl(3- 65-999 s Biohazards Control and Containment Program Segment.

Borrow., Pichard L. -- Collection of BurXitt African Lynphoma Specimens.'"'akerere University '.fedical School, Uganda. PH^3- 67 •1«7 3 Resources andLogistics Program Semnent.

^ioore, G. E. • Development of Methodology for Lar e cale In VitroPropagation of Human Leukemia Cells. Health Research, Inc., Buffalo.•/lew York F!'iU3»65~6l6, Devslopniental Research Prorran Segment.

1(9. '"'urphy ''. II.; Research on Virus Particles in Plasma and Tissue Cul-ture Specimens. University of ;fichican, Ann Arbor. Michigan. PF*t3-65 -639. Developmental Research Prograr Segment.

50. Oleson J. J.. Development of Virus-Cancer Test Systems- Virus Produc-tion- Production of Human Virus Cancer Cell Lines. Charles Pfizer anJCo.,, Inc. "aywood. New Jersey,, PHfc3-66--98, Developmental ResearchProe:rari Sepment.

51. Palczuk, N. C.: Iimunocheaiical and Antij^enicity Studies. RutgersUniversity New Brunsvick. Eew Jersey, PHl*3- 65-1039., DevelopmentalResearch Program Se rent.

52. Paliotta, A. J.- Virus Cancer Studies in Primates. Bionetics ResearchLabs... Inc., Kensington. Maryland, PM3-62-C1S, Developmental ResearchProgram Segnent.

53. Pledger, R.- Pauscher Leukenia Virus Production. Hazleton Labs., Inc.Falls Church Virginia, PBk3~65--635 Resources and Logistics ProgramSegment.

5**. Pollard 4 H, • Development of Leukemia Virus-Free, Gemfree i'ouse Colony.University of riotre Dame South Bend Indiana, PKJt-3-65-1018, Resourcesand Logistics Program Sepinent.

55. Read, R. B.: Studies on Thermal In&ctivation of Viruses in Milk ant? "ilkProducts. PUS Bureau of State Services (Taft Sanitary Enpineerinff Ctr.).Cincinnati. Ohio VCL 30. SALES Program Segment.

56. Reyniers J. A.• Comparative Studies of Carcinogen Activated ViralTumorigenesis in Germfree and Conventional Animals, Oermfree LifeResearch Center. TAir.pa Florida., PBl*3--65 95, Develourental ResearchProgram Seenent.

57. Rickard C.- Research on Susceptibility of Cat to Viral Leukemo^enesis.Cornell University. Ithaca, View York. PHU3-65 620^ SALJjS Program Sepnent.

58. Riopelle A. J.: Evaluation of Tree Shrew as Tumor Virus ResearchAnimal. Tulane University,. Covinpton Louisiana, PEU3-6U-881. Resourcesand Logistics Program Segnent.

59. Sorensen D, K.• Research on Susceptibility of Cows to Known TumorViruses. University of Minnesota, St. Paul, "innesota, PHU3-65-606,SALE- Program Segment.

60. Theilen G, H.: Bovine Leukenia 'I'r&nsmission Studies. University ofCalifornia Davis. California., PKU3-65 609, SALKS Prorrar Sepn>ent.

.1*6..

Gl, Verna. J.: Human Embryonic Tissue p.nd Cell Culture Facility CellBioloey Research, >'clear Inc., Falls Church Virpinia, PK^3-65 -6bResources and Logistics Proprap Sepnent,

62. T-"aravdek£rs V. S.- Research on J:ost Cell Virus Interrelationships.!r'icrobiolop;ical Associates, Inc., Bethesda "aryland; PH^3-66 887Proprram Management 3egr%ent.

63. "ilsnack R. Fluorescent Antibody Studies of Hurine Viruses.Baltimore Biological Lab., Inc., Baltimore, "aryland, FHl*3-63-ll6l.,Testing and I'onitorinp Program SeCTent.