A DD

>r-4

..... •TECHNICAL MANUSCRIPT 364

........... ,

INACTIVATION OF VENEZUELAN. . . . .:. . .EQUINE ENCEPHA' "MYELITIS VIRUS

: '.BY GAMMA ".. DIATION

*.....:

Morton Reitman....... :Henry R. Tribble, Jr.

...........

... ..... .. ...

............

•.........:.

APRIL 1967

. .his document is subject to spe iti a enrt ontrois and w n........ transmnittal to foreign rovern~met iA f oreiep 'ationals, my W#-: .- A. O-ly e y priRr apprrva i e o

• . ... .... c

-; :... ..... ,

DEPARTMENT OF THE ARMYFort Detrick

F.........

S..........

Reproduction of this publication in whole or inpart is prohibited except with permission of theComnanding Officer, Fort Detrick, ATTN: TechnicalReleases Branch, Technical Information Division,

Fort Detrick, Frederick, Maryland, 21701. However,DDC is authorized to reproduce the publication forUnited States Government purposes.

DDC AVAILABILITY NOTICES

Qualified requesters may obtain copies of thispublication from DDC.

Foreign announcement and dissemination of thispublication by DDC is not authorized.

Release or announcement to the public is notauthorized.

DISPOSITION INSTRUCTIONS

Destroy this publication when it is no longerneeded. Do not return it to the originator.

The findings in this publication are not to beconstrued as an official Department of the Armyposition, unless so designated by other authorizeddocuments.

_ B

DEPARTMENT OQ THE ARMYFort Detrick

Frederick, Maryland 21701

TECHNICAL MANUSCRIPT 364

INACTIVATION OF VENEZUELAN EQUINE ENCEPEALOMYELITISVIRUS BY GAMMA RADIATION

Morton Reitman

Henry R. Tribble, Jr.

" Medical Investigation DivisionM&DICAL. SCIENCES LABORA7ORY

andMedical Bacteriology Division

BIOLOGICAL SCIUMES UAOEATORY

Project 13622,601Al2............ .. April 1967WA Ii

I

2U

In conducting the research described in this report, theinvestigators adhered to the "Guide for Laboratory AnimalFacilities and Care," as promulgated by the Committee onthe Guide for Laboratory Animal Facilities and Care of theInstitute of Laboratory Animal Resources, National Academyof Sciences-Natioual Research Council.

ABSTRACT

Exposure of Venezuelan equine encephalomyelitis (VEE) virus atMO-70 C to 6 x 106 r gamma radiation (Co 60) resulted in loss of lethality

for young adult mice and guinea pigs and loss of capacity to produceplaques or cytopathic effects in tissue culture. The suckling mousewas more sensitive for detecting live virus in irradiated suspensionsthan the adult mouse or guinea pig. Live virus was demonstrable inpreparations exposed to 6 x 106 r but not in sispensions exposed to8 x 106 r or more, The rate of inactivation of VEE virus by gammaradiation was an exponential function of the dosage.

I

3

1 * INTRODUCTION

It has been reported that gamma rays inaccivate the viruses ofpoliomyelitis, St. Louis encephalitis, western equine encephalitis, vaccinia, 1

influenza, and mumps.O The results presented suggest that this method ofinactivation destroys the capacity of the virus to produce infectivityin animals but does not alter the antigenicity unless large doses ofradiation are employed. These studies prompted our investigation oninactivation of Venezuelan equine encephalomyelitis (VEE) virus hyexposure to gaama ray..

rmII. MATERIALS AND METHODS

A. VIRUS

The Trinidad sttain of VEE was used for all experiments. Virus waspropagated in monolayers of mouse fibroblast btrain L cells or in aMaitland-type culture of chick embryo tissue ý.n a chemically definedmedium. 3 Suptrnatants were harvested and clarified by centrifugation;portions were stored in sealed ampules or rubber-stoppered bottles at-70 C.

B. RADIATION

Virus suspensions were radiated at the National Bureau of Standards,using a 50,000-curie cobalt 60 source emitting radiation at a rate ofapproximately 7.5 x 106 r per hour. Cobalt glass dosimetry was used tomeasure the exact dosage delivered. Suspensions were irradiated in glasscontainers sealed in tin cans in a Dewar flask and inserted into a carrier(Fig. 1). The samples were kept frozen with dry ice during exposure tominimize the indirect lethal action of free radicals.4 After exposure,samples were stored at -70 C until tested for presence of active virus.Control samples of virus were exposed to identical conditions but were notirradiated.

C. TITRATION OF VIRUS SUSPEWSION

Survival curves of irradiated virus were determined by intracerebral(IC) inoculation of 0.03 ml in 10- to 14-g Swiss mice and by plaque

strain L cells. Samples of irradiated virus that were nonlethal for adultmice were inoculated into 250- to 350-g guinea pigs. Each of 15 guineapigs was administered 0.25 ml of an undiluted or a 10-1, 10-2, 10-3, or10-4 dilution of virus. No deaths occurred.

-•- _ _ _ _---__ _ _--

3"9-

Carrier

Dry Ice

12" Dewar Insert--- Tin Can

Sample(serum bottle or vial)

II

Figiure 1. Container Arrauguamt for irradiating Samples.

II

I _ _ _ __ _

5

On the basis of data obtained, guinea pigs (250 to 350 g) wereinoculated with selected samples of irradiated VEE virus. These samplesranged from those exposed to doses of radiation that failed to inactivateall viable virus to those that were nonlethal to adult mice and guineapigs. The animals received either one or two inoculations given at aninterval of I week; dosages are given in Table 3. Five animals werebled 3 weeks postinoculation by intracardial puncture arid the level ofserum-neutrallzing and hemngglutination-inhibiting antibodies wasdetermined. The resistance of the guinea pigs to challenge with virulenthomologous virus was determined. A dose of 2.5 x 105 mouse IC lethaldoses50 (ZAICLD) wac administered to each animal. Mice were utilized insimilar tests but no serologLcal assays were performed.

Virus suspcLub.uas were titrated in suckling mice by inoculation of0.03 ml via the IC and 0.03 ml via the intraperitoneal (LP) route in thesame animal. The brains of mice that died 24 or more hours afterinoculation were harvested, ground in a mortar, and suspended in beefheart infusion broth. The presence of VEE virus was confirmed byneutralization with VEE antiserum in adult mice.

D. SERUM NEUTRALIZATION TEST

The serum neutralization (SN) test reported by Smith et al.5 wasemployed, utilizing male mi'e only.

E. HEMAGGLUTINATION INHIBITION TEST

Guinea pig sera were tested by the microtiter technique using a VEEhemagglutinin antigen inactivated with beta-propiolactone.

III. RESULTS

Figure 2 shows the effect of various doses of ga--- radiation on thesurvival of VEE virus as determined by assay in adult mice. The medianlethal dose (LDO values, calculated according to the Reed and Muenchformula,6 are plotted on a logarithmic scult as a function of radiationdose. The results show that the rate of inactivation is expressed as anapproximately straight line, indicating that the surviving fraction is anexponential function of the dose. Extension of the line indicates thatcomplete loss of mouse lethality could be expected to occur at a doselevel of 7.4 x 106 r. However, deaths did not occur when younlg adult micewere inoculated with virus that had been exposed to radiation doses of6 x 106 r or greater (Table 1). Similar results were obtained with guineapigs (Table 2). The capacity to produce plaques or cpe was lost insuspensions exposed to 6 x 106 r or greater.

ii

6

876

5

4-0

S3 -

E

1 2 -

0%

106 x Roentgens

Figure 2. Survival Curve of VEE Virus Obtained by Irradiationwith Gaman Rays, Average of Tw~o Experiments.

I!'

i

The results of the assays in suckling mice and the hemagglutinationinhibition (HI) and SN tests from a typical experiment are shown in Table 2.The test with suckling mice indicated the presence of live virus in thesuspensions irradiated with 5 x 106 r and 6 x 106 r, but not in thosesuspensions exposed to larger doses. The virus exposed to 5 x 106 r waslethal for 207. of guinea pigs but not for adult mice. High SN indiceswere obtained in all guinea pigs administered two inoculations of virusthat had been exposed to radiations ranging from 5 x 106 to 10 x 106 r.A significantly lower SN antibody response was obtained in guinza pigsinoculated with virus exposed to 16 x 106 r. Assuming that virulence forsuckling mice is correlated with the amount of live virus present in theirradiated suspensions, the lowest HI antibody responses were obtainedwith the preparation containing the greatest amount of live virus. Theprotective potency d-termined by antigen extinction assay is expressedhere as the effective dose (EDO), the volume of undiluted irradiatedvirus suspension inoculated per dose that protected 507. of the testanimals. The ED. for mice was the same for preparations exposed toradiation doses ranging from 5 x 106 r to 8 x 106 r but was greater athighec radiation levels.

Table 3 lists the SN antibody response and the ED3c in millilitersobtained in guinea pigs that received either one or two inoculations ofirradiated virus. The primary antibody response was low in the animalsthat received only one inoculation. The second inoculation caused anincrease in antibody in 507. of the animals. It should be noted that Tables2 and 3 list the results obtained by inoculation of two different viruspreparations and that the guinea pigs listed in Table 2 received 0.25 mlmore virus than those listed in Table 3.

TABLE 1. INACTIVATION OF ViE VIRUS BY GAMMA RADIATION AS MEASURED BYPLAQUE FORMATION, cpe, AND M)USE LETHALITY./

RadiationDose, 106 r MICLD5 pfub/ cpeh/

0 10.0 8.3 8.3

1 7.6 6.0 7.5

3 4.1 2.8 3.56 <1.5 <1.0 <1.0

10 <1.5 <1,0 <P. 0a. Experiment HV-l, L cell culture. MICLDW~ml 10.0 prior to

radiation.Log Lase 10.

b. Performed by Dr. Henry J. Hearn, Jr.

_______________________________________________________________

.0 . %0 %a 0 0 0

VZ A A~ A~~9~' ~'al 4 I .1Go 1-4 4

f-4 cl t au% d

P. %0 goe j

100U0

r-4 4.1 0-4

1- . a

V. 0 4)

1-4 w

0-40 a -

-4 $4 +4

00-4

-4 C4c

C4~~ a Aj1

E~~~U-4 c J40 .Z

.-4 u 04j U 4 014404 lo o X -

0-4

1. U1171

4) '9.4A'

414cc1

C/3 4J-4

J.I 00 O 0 OO5~z- 00 0 0N Cna%-4-4H 0bOc 0. N

o- cc a Q 0

41

1. .4!

.I 4!.1

C,-

4! 4

-44

000

4r4

I

10

IV. -DISCUSSION

The results of tests fir inactivation of VEE virus by measuring thecapacity of the virus to produce epe, plaques, or death of adult mice and

- guinea pigs suggest that active virus was not present in suspensions exposedto 6 x 10 r of gamma :adiation. However, live virus was detected in this

- preparation by inoculation of suckling mice by brth the IC and IP routes.Suckling mice have been reported to be considerably more sensitive to infectionwith VEE virus than either hamster kidney or monkey kidney tissue cells.'

- It is apparent from our experiments that the suckling mouse is more sensitiveto the lethal action of irradiated VEE virus than either the adult mouse orguinea pig.

The use of ionizing radiation to inactivate virus presents the difficult- problem of determining the absence of live virus in irradiated preparations

because the survival curve of ihradiated viruses is exponential.'

S •The high levels of SN and HI antibodies obtained in the guinea pig(Table 2) might be due either to an inapparent infection with live virus or3 to the stimulation of antibody production upon introduction of additionalinactivated virus in the second inoculation. The latter appears to be themore logical explanation. The data listed in Table 3 show that oneinoculation was not sufficient to induce significant antibody titers; asecond inoculation resulted in a sharp rise in the level of neutralizingantibody. These results also support the conclusion that live virus was notpresent in sufficient quantity to produce infection, because it would beexpected that significant levels of antibody would have resulted from asingle inoculation.

The reduced ancibody response that oL..rred at radiation doses of 10 x 106 ror higher may be attributable to breakdown of antigenic material; thisphenomenon has been reported to occur with the viruses 6f influenza andvaczinia.8

-z• V. SUMWARY

A strain of VEE virus was inactivated by exposure to a radiation doseS I of 8 x 106 r of gamma radiation. The suckling mouse was more sensitive for

detecting live virus in irtadiated suspensions than the adult mouse or guinea* pig. The rate of inactivation of VEE virus by gamma radiation was an

exponential function of the dosage.I

Ii

|-IL

I _ __ __ __ ___ _ _ _ _ _ _ _

•'• l l l l l l l l l l l l l l I

"LTEMAUS CITED

1. Jordan, R.I.; Kempe, L.L. 1956. Inauctivation of some animal viruseswith gamma radiation from cobsalt 60. Proc. Soc. Exp. Biol. Med.9 1:212-215.

2. Polley, J.R. 1962. The use of gamma radiation for the preparation

of virus vaccines. Can. J. MLcrobiol. 8:455-459.

3. Nagle7ýS.C.; Tribble, H.R.; Anderson, I.E.; Gary, N.D. 1963.A chemically defined medium for growth of animal cells in suspension.Proc. Soc. Exp. Biol. Med. 112:340-344.

4. Sykes, G. 1965. Disinfection and sterilization. 2nd ed. E & F SponI..' Ltd., London. 486 p.

5. Suaith, D.C.; Mamay, U.K.; M~arar., 1.0.; Wagner, J.C. 1956.Venezuelan equinu encephalamyelitis: Laboratory aspects of fourteenhuman cases following vaccination and attempts to isolate the virusfrom the vaccine. Amer. J. Hyg. 63:150-164.

6. Reed, L.J.; Muench, HI. 1938. A simple method of estimating fifty per centendp6ints. Amer. J. Hyg. 27:493-497.

7. IXudin,, W.D.; Liu, C.; Rodina, F. 1966. Pathogenesis of Venezuelanequine encephalomyelitis virus: I. Infection in subkling mice.J. Immunol. 96:39-48.

8. Polley, J.R. 1961. Factors influencing inactivation of infectivityand hemagglutinin of influenza virus by gamma radiation. Can. J.Microbiol. 7:535-541.

9. Kaplan, C. 3.960. The antigenicity of .yrirradiated vaccinir, virus.J. Hyg. 58:391-398.'

UnclassifiedSecurity Classification

DOCUMENT CONTROL DATA' R&D(0.OC tly 0I6.delica1ial i. l.9 ., b. ip#ir =n Ind.mi*R mlai o N.t . sneered thll- the o-alI -,.I I pl .. )lteL*,d)

I. ORIGINIATING ACTIVITY (Cwtesetm. -0-ho1) 120. REPORT S9CLURITy C LAlS*IICAJTION

Department of the Army UnclassifiedFort Derrick, Frederick, Maryland, 21701 2b CROuP

3. REPORT TITLE

INACTIVATION OF VENEZUELAN EQUINE ENCEPHALONYELITIS VIRUS BY GAMMA RADIATION

4 DESCRIPTIVE NOTES fT,,.1 aN •,p and Jnlusve dv.ole*)

S. AUTHORIS) (LM .( nmme, firs .as. mintial)

Reitman, Morton (NMI)Tribble, Henry R., Jr.

6. REPORT DATE 7.. TOTAL NO. OF IA;Ils 7b. NO O REmSApril 1967 14

S4a. C€ONTRACT OR GRANT 14O. 9A. ORIGINATONWS REPORT NUMOBER(S)

.0" I RO.,CT NO. IB622401A072 Technical Manuscript 364

€., .18 1rPOeRY NO(S) (Any oth., numesb. A.... bo nh.. 1ted

I . A VA IL. AILITY/LIMITATIOMN NOTICESQualified requesters mp, !-htain copies of this publication from DDC.Foreign announcement a: tIb-. iation of this publication by DDC is not authorized.Release or announcement o the public is not authorized.

I I. SU PPL. EMENTARY NOTES 12- SPONSORING MILITARY ACTIVITYDepartment of the ArmyFort Detrick, Frederick, Maryland, 21701

S. ABSTRACT

Exposure of Venezuelan equine encephalomyelitis (VEE) virus at -70 Cto 6 x 106 r gamma radiation (Co 6 0 ) resulted in loss of lethality for youngadult mice and guinea pigs and loss of capacity to produce plaques or cytopathiceffects in tissue culture. The suckling mouse was more sensitive for detectinglive virus in irradiated suspensions than the adult mouse or tuinea pig.Live virus was demonstrable in preparations exposed to 6 x 10 r but-not insuspensions exposed to 8 x 106 r or more. The rate of inactivation of VEEvirus by gamma radiation was an exponential function of the dosage.

14. Key WordsVenezuelan equine encephalitisRadiationMice -

Guinea pigsPlaquesTissue culturesSuspensionInactivation

* I0D ,o2D 1473 Unclassified

Security Classificatioa

ii