Responses of Isolator-Derived Japanese Quail and Quail Cell Cultures to Selected Animal Viruses

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1975, p Copyright C) 1975 Americn Society for Microbiology Vol. 2, No. 5 Printed in U.SA. Responses of Isoltor-Derived Jpnese Quil nd Quil Cell Cultures to Selected Animl Viruses WENDALL M. FARROW,* MARTIN W. SCHMITT, AND VINCENT GROUPE Life Sciences Reserch Lbortories, Life Sciences, Inc., St. Petersburg, Florid Received for publiction 11 July 1975 Thirteen oncogenic nd necrotizing niml viruses were ssyed in Life Sciences, Inc. (LSI)-specific pthogen-free Jpnese quil nd LSI-specific pthogen-free chicken embryo cell cultures. Nine viruses produced similr titers in the quil nd chicken cell systems, wheres four viruses showed significntly higher titers in chicken. Young Jpnese quil nd chickens were inoculted with five selected vin viruses nd mintined in stinless-steel isoltors. Comprble responses were noted in quil nd chickens injected with Newcstle disese virus nd vin leukosis virus, but quil were significntly more resistnt thn chickens to fowl pox virus, lryngotrcheitis virus, nd Mrek's disese herpesvirus. Although no overt symptoms of disese were observed in Jpnese quil inoculted with most vin viruses, neutrlizing ntibody or virus ws detected, indicting presence of n inpprent infection. In one experiment, neutrlizing ntibody ws detected in comprble number of quil nd chickens fter inocultion with vin leukosis virus. Avin leukosis virus viremi ws observed t 12 nd 70 dys postinocultion, with the COFAL (complement fixtion for vin leukosis) titers similr for quil nd chickens. Most quil infected with Mrek's disese herpesvirus produced neutrlizing ntibody within 70 dys but showed no clssicl symptoms of Mrek's disese even when held for 5 months. In contrst, ll chickens inoculted with Mrek's disese herpesvirus died within 20 dys. The utility of quil embryo cell cultures in the preprtion of vccines nd biologicl regents is discussed. Domesticted Jpnese quil (Coturnix coturnix jponic, Temminck nd Schlegel) re useful in biologicl reserch becuse of their smll size, erly mturity, nd high egg productivity (14, 21). Coturnix produce fertile eggs s erly s 35 dys nd mintin reltively high fertility rte (70 to 80%) for period of pproximtely 7 months. Eggs, which re usully speckled, htch in 16 to 17 dys, nd dult birds verge 95 nd 125 g for mles nd femles, respectively. Within brrier environment, Jpnese quil live up to 5 yers, which is equivlent to the longevity of chickens. Previous studies (12, 15, 25-27) hve demonstrted the vried responses of conventionl Coturnix to inocultion with certin vin tumor viruses, wheres quil embryonted eggs (10, 17) were found to support the growth of severl viruses. Additionlly, Jpnese quil cell cultures hve been utilized in studies on the leukosis-srcom group of viruses (8, 9, 25, 29, 30, 32) nd certin herpes-type viruses (2, 13, 16). Quil embryo cells were essentilly resistnt to subgroup B virus (31) nd showed reduced susceptibility to subgroup C viruses (6). Since 1967 we hve developed nd mintined rigidly monitored (7), specific pthogen-free (SPF), outbred flock of Jpnese quil. The flock, derived from n isoltor environment, is free of exogenous vin leukosis virus(es) (ALV), Mrek's disese herpesvirus (MDHV), nd other conmmon vin virl nd bcteril pthogens. Definitive (germfree, SPF) Coturnix hve been used for studies (1, 18, 21) only on selected Rous srcom viruses [RSV, RSV(O)]. Therefore, it ws importnt to extend studies to determine the response of SPF quil nd quil cell cultures to common vin viruses nd to certin mmmlin viruses. ALV nd MDHV were included becuse of the importnce of estblishing source of cells free of ALV nd MDHV. MATERIALS AND METHODS Viruses. CELO virus, fowl pox virus (FPV), herpes simplex virus, nd vesiculr stomtis virus t high pssge levels in chicken eggs were pssged n dditionl two to three times in chicken embryo secondry cells (CEF). Influenz (WSN-125) virus nd the Rokin strin of Newcstle disese 419

2 420 FARROW, SCHMITT, AND GROUPE virus (NDV) were prepred from infected llntoic fluids fter seven pssges in chicken embryonted eggs. Lryngotrcheitis virus (LTV) ws pssged four times on chicken embryo choriollntoic membrnes. All of the bove viruses were obtined from J. W. Frnkel, Life Sciences, Inc., St. Petersburg, Fl. The Beudette strin of infectious bronchitis virus (IBV) ws obtined from C. H. Cunninghm, Michign Stte University. The virus ws propgted in chicken eggs nd then pssged mny times in chicken kidney cells (CK) (4); three dditionl pssges were mde in CK in this lbortory. The MDHV (GA isolte) ws supplied by C. Eidson, University of Georgi, nd the MDHV inoculum ws derived from skin nd fether tips of resistnceinducing fctor, Life Sciences, Inc. (LSI)-SPF chickens. ALV-42 ws received s low-pssged (two to three times) CEF preprtion from R. M. Dougherty, Stte University of New York t Syrcuse. The Rous srcom pseudotypes, RSV(RAV-1) nd RSV(RAV-6), were supplied by P. Meyers, Myo Medicl School t Rochester, nd virl stocks were prepred t the second pssge in CEF. Preprtions of the Bryn high titer RSV, derived from chicken tumor, nd RSV(RAV-50) were obtined from the Resources nd Logistics Segment, Virus Cncer Progrm, the Ntionl Cncer Institute, nd pssged two times in CEF. Cell cultures. Quil embryo secondry cells (QEF) nd CEF were prepred from 5- to 6-dy-old primry fibroblsts derived from 7- to 9-dy-old embryos. Cells were trypsinized nd seeded in 60- mm plstic petri dishes t concentrtion of 4.0 x 105 cells/plte. Growth medium ws Egle minimum essentil medium (MEM) prepred with Erle slts, supplemented with double-strength vitmins nd glutmine, 10% het-inctivted fetl clf serum (FCS), 10% tryptose phosphte broth, nd ntibiotics. Cultures of QEF nd CEF were incubted t 37 C, nd cell monolyers were confluent within 48 h. Quil primry cells (QK) nd CK, prepred from 1-dy-old birds nd seeded t 8.0 x 105 cells/plte in MEM, formed confluent monolyers in 72 h. To determine virl infectivity titers, confluent cell monolyers were inoculted with 0.5-ml volumes of virus suspension nd bsorbed for 1 h t 37 C. The overly medium (MEM) contining 5% FCS nd 0.4% gr (Difco) ws dded, nd infected cell cultures were observed for cytopthic effect (CPE) during period of 9 dys. For titrting RSV nd RSV pseudotypes, the virus suspension ws inoculted into the growth medium, n gr overly ws dded t 18 h, nd two dditionl overlys were dded 3 dys prt. Polybrene (Aldrich Chemicl Co., Milwukee) ws dded to the diluent t concentrtion of 10,ug/ml to enhnce the bsorption of RSV(RAV-6) nd RSV(RAV-50). MDHV ws bsorbed for 30 min t 37 C in MEM contining 0.05% Versene nd 2% FCS, nd fresh medium without Versene ws dded t 24 h. When the medium ws chnged 18 h lter, 0.4% gr (Difco) ws included nd FCS ws reduced to 2%. Foci were recorded 7 dys lter. Neutrliztion tests. Quil nd chicken ser were inctivted t 56 C for 30 min nd diluted 1:20 J. CLIN. MICROBIOL. in MEM. The serum dilution ws mixed with n equl volume of virus suspension nd incubted for 30 min t room temperture prior to inocultion of CK or CEF. The virus control contined diluent in volume equivlent to the virus suspension. After inocultion, the mixture ws bsorbed for 30 min t 37 C, n overly ws dded, nd the number of foci ws counted 8 dys lter. A positive neutrlizing ntibody (NA) response ws recorded when 90% or greter reduction in focus-forming units or plqueforming units ws observed. Complement fixtion for vin leukosis (COFAL) test. The complement fixtion test ws performed using the microtiter technique (24). The ntiserum used to detect COFAL ntigen (23) ws obtined from hmsters inoculted with the Schmidt-Ruppin strin of RSV. Whole blood collected from fsted birds ws diluted 1:5 in cold sline (0.85%) nd dispensed in 0.5-ml volumes to plstic petri dishes seeded with CEF. MEM supplemented with 10% FCS ws dded, nd cells were split every 5 to 7 dys during period of 3 weeks. Cell cultures were frozen nd thwed twice prior to testing for COFAL ntigen. Group-specific ntigen ws lso determined in quil embryo livers derived from 14-dy-old embryonted eggs (5). Birds. The LSI-SPF Coturnix used in this study were derived from closed outbred flock mintined t the Germfree Life Reserch Center, Tmp, Fl. Fertile eggs obtined from this flock were surfce disinfected nd htched in sterile Reyniers stinless-steel isoltor (19). Newborn quil were rered for pproximtely 30 dys in the isoltor environment nd then trnsferred septiclly to brriersustined cubicles (20) for expnsion of smll breeder flocks. LSI-SPF White Leghorn chickens were derived from flock developed t the University of Connecticut by R. Luginbuhl. Selected, fertile eggs collected from trp-nested chickens over period of severl months were surfce disinfected nd htched in the sme mnner s described for quil eggs. The chickens were rered to 2 weeks of ge nd lter developed s pedigreed flock in primry brrier system. Monitoring of both SPF quil nd chicken flocks for vin pthogens followed estblished procedures (11). Serum neutrliztion nd hemgglutintioninhibition tests were used to detect ntibodies to ALV, CELO virus, FPV, IBV, LTV, MDHV, nd NDV. Slide gglutintion tests were performed for Mycoplsm gllisepticum, M. synovie, nd Slmonell pullorum. No detectble vin pthogens were observed in these monitoring tests. Additionlly, COFAL (23) nd resistnce-inducing fctor (22) tests were repetedly negtive. RESULTS Infectivity titers in cell culture. The titers of 13 oncogenic nd necrotizing niml viruses in Jpnese quil nd chicken cell cultures re shown in Tble 1. The infectivity titer of RSV (Bryn) ws 1 log lower in QEF thn in CEF, nd similr response ws observed with

3 Viru) Virus DoePFUb I:ocllltiSymptomscMortlityc VOL. 2, 1975 RSV(RAV-6). With the pseudotypes RSV(RAV- 1) nd RSV(RAV-50), there ws no significnt difference in titers in the quil nd chicken cell systems. The titer of MDHV in QK nd CK ws comprble, lthough the time required to obtin distinct foci ws 2 dys longer in the quil system. Two other vin viruses, FPV nd NDV, gve slightly higher titers in QEF. CPE ws detected within 72 h in QEF nd CEF infected with FPV nd 24 h fter inocultion with NDV. CELO virus, IBV, LTV, nd MDHV were ssyed in kidney cells since CPE ws more distinct thn in embryo cell culture. The titers of CELO virus nd LTV were significntly lower in QK. CPE ws detected within 48 h in CK inoculted with CELO virus, IBV, LTV, nd MDHV, wheres in QK the viruses showed no CPE erlier thn 72 h. The infectivity titers of herpes simplex virus nd vesiculr TABLE 1. Virus titers in cell cultures derived from LSI-SPF quil nd from chickens Cell Log infectivity titer Virus culture (FFU or PFU/mlP type Quil Chicken RSV (Bryn) Embryo RSV (RAV-1) Embryo RSV (RAV-6) Embryo RSV (RAV-50) Embryo FPV Embryo NDV Embryo Herpes simplex Embryo Vesiculr stomti- Embryo tis Influenz (WSN) Embryo MDHV (GA) Kidney CELO Kidney IBV Kidney LTV Kidney FFU, Focus-forming units; PFU, plque-forming units. QUAIL CELL CULTURE RESPONSES TO VIRUS 421 stomtitis virus were identicl in QEF nd CEF, with cell cultures showing CPE within 24 h nd mximum effect by 48 h. The virus titer of WSN ws 4 logs lower in QEF thn in CEF. Mximum CPE ws observed within 48 h in CEF nd by 96 h in QEF. Response of LSI-SPF Jpnese quil inoculted with selected vin viruses. LSI-SPF Coturnix nd chickens, htched nd mintined in n isoltor environment, were inoculted simultneously t 3 dys of ge with the vin viruses shown in Tble 2 nd observed for morbidity nd mortlity for period of 10 weeks. Quil inoculted with FPV or LTV showed no overt symptoms chrcteristic of disese, wheres ll infected chickens developed typicl responses 4 to 7 dys postinocultion, showing vrible mortlity rte of 27 to 60%. The verge dy of deth ws 10 for chickens inoculted with FPV nd 12 for those injected with LTV. Liver nd spleen extrcts were prepred from FPV- nd LTV-inoculted quil nd chickens held for 10 weeks. When 10% suspensions of these extrcts were injected onto the choriollntoic membrne of 9-dy-old embryonted chicken eggs, numerous pocks typicl of FPV nd LTV were detected fter 6 dys of incubtion. The response of Coturnix nd chickens inoculted with NDV ws similr lthough some difference ws observed in time of deth; the disese progressed rpidly in chickens nd ll injected birds died within 48 h, wheres quil survived up to 96 h. Quil nd chickens inoculted with MDHV showed mrked vritions in response. None of the chrcteristic symptoms of Mrek's disese ws detected in Coturnix during period of 10 weeks, nor were clssicl viscerl nd neurl tumors found in tissues of birds. However, NA ws detectble in serum smples of five rndomly selected MDHV-inoculted quil t 10 weeks. The response in LSI-SPF chickens ws TABLE 2. Responses of LSI-SPF Jpnese quil nd chickens inoculted with vrious vin viruses Inocultion Cumultive responses Dose (FFU or ~~~Quil Chickens or Route Symptoms Mortlityc FPV 10 Comb scrifiction LTV 10 Intrtrchel NDV (Rokin) 10 Intrperitonel MDHV (GA) 10 Intrcerebrl ALV 10 Intrperitonel Groups of 15 birds held for 70 dys. b FFU, Focus-forming units; PFU, Plque-forming units. c Percentge.

4 422 FARROW, SCHMITT, AND GROUPE similr to tht seen erlier (7). The mortlity rte ws 100%, nd ll chickens died 12 to 18 dys fter injection with MDHV. No gross symptoms of infection were detected in quil nd chickens inoculted with ALV. In contrst, kidney, liver, nd spleen homogentes derived from ALV-inoculted quil nd chickens did show COFAL titers of 1:4 to 1:8 fter pssge in CEF for 18 dys. Antibody response of LSI-SPF Jpnese quil nd chickens inoculted with ALV or MDHV. In one experiment, 3-dy-old Coturnix nd chickens, htched nd mintined in n isoltor brrier, were inoculted intrperitonelly with 100 men tissue culture infective doses of ALV or with pproximtely 200 focusforming units of MDHV. Ten weeks fter inocultion serum smples were collected from ech bird nd NA ws determined. NA to ALV ws detected in 12 of 15 quil nd in slightly higher number (14/15) of chickens (Tble 3). The NA response in MDHV-inoculted LSI- SPF quil nd chickens could not be compred since ll chickens died 12 to 20 dys fter infection. In one group of 15 MDHV-inoculted quil, 11 showed detectble NA 10 weeks fter injection. Similrly, 6 of 8 birds held for 5 months contined MDHV ntibody. Quil dem- TABLE 3. Detection ofalv-ntibody in serum from isoltor-held Jpnese quil nd chickens 10 weeks fter inocultion with ALV Host Inoculum No. with ntibody/totl Quil ALVb 12/15 Quil Diluent 0/15 Chickens ALV 14/15 Chickens Diluent 0/15 1/20 serum dilution effecting 90% or greter reduction with 102 focus-forming units of RSV (RAV- 1). b 100 men tissue culture-infective doses per bird. TABLE 4. J. CLIN. MICROBIOL. onstrting positive ntibody response showed no gross symptoms of Mrek's disese when necropsied fter n extended holding period of 5 months. ALV-viremi nd COFAL ntigen responses in LSI-SPF quil nd chickens. Whole blood smples were collected from ALVinoculted Coturnix nd chickens t 12 nd 70 dys. The blood ws inoculted in CEF, nd the cells nd fluids were tested for presence of COFAL ntigen t 21 dys, using the microtiter complement fixtion test. Viremi ws detected in quil nd chickens t 12 dys nd ws persistent in birds bled t 70 dys (Tble 4). The rnge of COFAL titers ws similr in quil nd chickens t 12 nd 70 dys postinocultion. The titers of COFAL ntigen were reltively low t 12 dys but incresed significntly during the course of the experiment. The number of quil responding to inocultion with ALV ws somewht less thn chickens. Beginning with the egg lying period t pproximtely 45 dys, eggs were rndomly collected from ALV-inoculted Coturnix nd tested for presence of verticlly trnsmitted COFAL ntigen. Eggs were incubted 7 to 9 dys, nd QEF cell cultures were prepred. After three pssges t 7-dy intervls COFAL ntigen titers were determined. Titers of 1:4 to 1:8 were obtined in pproximtely 40% of 30 quil eggs. Similr COFAL ntigen titers were lso demonstrted in extrcted single quil embryo livers derived from 13- to 14-dy-old embryonted eggs. DISCUSSION Although the titers of few viruses were lower in quil embryo cell culture thn in quil embryonted eggs (17), most of the viruses produced equivlent titers in the two systems. Additionlly, the titers of number of viruses in LSI-SPF Jpnese quil cell cultures were quite similr to those obtined in the LSI-SPF ALV-viremi nd COFAL ntigen response in LSI-SPF quil nd chickens fter intrperitonel inocultion with ALV ALV-viremi Reciprocl of COFAL titer Host Inoculum Rnge Geometric men 12 dys 70 dys 12 dys 70 dys 12 dys 70 dys Quil ALVb 12/15 12/ Quil Diluent 0/15 0/15 <2 <2 <2 <2 Chicken ALV 14/15 15/ Chicken Diluent 0/15 0/15 <2 <2 <2 <2 COFAL tests; frctions denote number of birds positive/number inoculted. b Approximtely 100 men tissue culture infective doses per bird.

5 VOL. 2, 1975 QUAIL CELL CULTURE RESPONSES TO VIRUS 423 chicken system. However, notble exceptions were CELO virus, IBV, nd WSN virus, which produced substntilly lower titers in quil cells. The pssge history of these viruses in the chicken system my ccount for the mrked differences observed. In study (3) with IBV, some degree of dpttion to growth in chicken eggs ws found essentil before the virus ws dpted to tissue culture, suggesting tht this procedure my be necessry to chieve comprble titers with CELO virus, IBV, nd WSN virus in quil nd chicken cell cultures. The bsence of overt symptoms of illness nd mortlity in LSI-SPF Coturnix inoculted with FPV, LTV, nd MDHV indicted greter resistnce to these vin pthogens thn ws observed with the LSI-SPF chickens. The response ws not ltered in quil held for n extended period fter inocultion, wheres LSI- SPF chickens responded to the viruses within norml incubtion time (4 to 18 dys). The holding time for ALV-inoculted quil nd chickens ws probbly insufficient s indicted by the bsence of illness nd gross lesions in chickens. In this experiment, detectble ntibody or virus ws found in serum smples or orgns (kidney, liver, spleen) of surviving SPF quil nd chickens inoculted with ALV, FPV, LTV, nd MDHV, indicting tht inpprent infection did occur. Only with NDV did SPF Jpnese quil nd chickens show comprble susceptibility (100%) nd mortlity (87, 100%). It is conceivble tht incresing the dose of virus nd using virus preprtions derived from the quil system might lter the response of SPF quil to some of the viruses. In one experiment, where the dose level of ALV ws incresed, LSI-SPF Coturnix nd chickens still did not show overt symptoms or gross tumors during period of 10 weeks. However, NA ws detected in 80% of inoculted quil, which ws similr to the response observed in chickens (93%). Moreover, the SPF Jpnese quil nd chickens were similr in the number of birds showing viremi nd in the titers of COFAL ntigen detected t 12 nd 70 dys postinocultion. In SPF quil inoculted with higher dose of MDHV, the ntibody response ws similr to tht of quil injected with ALV. Similr results were lso noted in the lck of tumor development nd bsence of mortlity. The utility of SPF Jpnese quil nd cell cultures in virus reserch is demonstrted by their responses to vriety of oncogenic nd nononcogenic viruses. Vccine viruses could conceivbly be produced in quil cell cultures, in which endogenous oncornviruses hve not been identified. Also, vriety of dignostic virl nd serum regents might lso be prepred in quil nd quil cell cultures. ACKNOWLEDGMENT This study ws conducted under Public Helth Service contrcts N01-CP nd N01-CP within the Virus Cncer Progrm. LITERATURE CITED 1. Bryn, W., M. R. Scksteder, R. D. Schwrtz, J. P. Kvedr, P. K. Vogt, nd J. Wrren Reltionship beween inititing dose nd concentrtion of Rous srcom virus (Type 0) in tumors of germfree quil. J. Ntl. Cncer Inst. 46: Colwell, W. M., D. G. Simmons, nd K. E. Muse Use ofjpnese quil embryo fibroblst cells for propgtion nd ssy of turkey herpesvirus FC-126. Appl. Microbiol. 27: Cunninghm, C. H Avin infectious bronchitis. Adv. Vet. Sci. Comp. Med. 14: Cunninghm, C. H., nd M. P. Spring Some studies of infectious bronchitis virus in cell culture. Avin Dis. 9: Dougherty, R. M., nd H. S. DiStefno Lck of reltionship between infection with vin leukosis virus nd the presence of COFAL ntigen in chick embryos. Virology 29: Duff, R. G., nd P. K. Vogt Chrcteristics of two new vin tumor virus subgroups. Virology 39: Frnkel, J. W., W. M. Frrow, C: 0. Prickett, M. E. Smith, W. F. Cmpbell, nd V. Groupe Responses of isoltor-derived nd conventionl chickens to Mrek's disese herpesvirus nd vin leukosis virus. J. Ntl. Cncer Inst. 52: Freemn, G Focus formtion by Jpnese quil cells infected with Rous srcom virus. J. Ntl. Cncer Inst. 31: Friis, R. R Abortive infection of Jpnese quil cells with vin srcom viruses. Virology 50: Higgins, D. A Growth of Newcstle disese virus in fertile eggs of the Jpnese quil (Coturnix coturni.xjponic). J. Comp. Pthol. 82: Methods for exmining poultry biologics nd for identifying nd quntifying vin pthogens Committee on Animl Helth, Ntionl Reserch Council, Wshington, D.C. 12. Moscovici, C., nd E. H. McIntyre Effect of vin myeloblstosis virus in the Jpnese quil. J. Bcteriol. 92: Onod, T., K. Ono, T. Konobe, M. Nito, Y. Mori, nd S. Kto Propgtion of herpes type virus isolted from chickens with Mrek's disese in Jpnese quil embryo fibroblst. Biken J. 13: Pdgett, C. A., nd W. D. Ivey Coturnix quil s lbortory reserch niml. Science 129: Pient, R. J., nd V. Groupe Growth curve nd distribution of Rous srcom virus (Bryn) in Jpnese quil. J. Virol. 1: Purchse, H. G., nd B. R. Burmester Responses of cell cultures from vrious vin species to Mrek's disese virus nd herpesvirus of turkeys. Am. J. Vet. Res. 32.: Ruscher, F. J., J. A. Reyniers, nd M. R. Scksteder Jpnese quil egg embryo s host for viruses. J. Bcteriol. 84: Ruscher, F. J., J. A. Reyniers, nd M. R. Scksteder Responses or lck of response of pprently leukosis-free Jpnese quil to vin tumor viruses, p In Monogr. 17. Int. Conf. Avin Tumor Viruses, Duke Univ., Durhm, N.C.

6 424 FARROW, SCHMITT, AND GROUPE 19. Reyniers, J. A Design nd opertion of pprtus for rering germfree nimls. Ann. N.Y. Acd. Sci. 78: Reyniers, J. A Controlled environmentl fcility for mintining closed niml qurters. Lb. Anim. Cre 14: Reyniers, J. A., nd M. R. Scksteder Rising Jpnese quil under germfree nd conventionl conditions nd their use in cncer reserch. J. Ntl. Cncer Inst. 24: Rubin, H A virus in chick embryos which induces resistnce in vitro to infection with Rous srcom virus. Proc. Ntl. Acd. Sci. U.S.A. 46: Srm, P. S., H. C. Turner, nd R. J. Huebner An vin leucosis group-specific complement-fix-' tion rection: ppliction for the detection nd ssy of non-cytopthogenic leucosis viruses. Virology 23: Sever, J. L Appliction of micro-technique to virl serologicl investigtions. J. Immunol. 88: Shipmn, C., Jr., nd A. V. Levine Tumor production in the Jpnese quil by the Schmidt-Ruppin strin of Rous srcom virus. Virology 27: Smid, J., nd V. Smidov Preliminry report. J. CLIN. MICROBIOL. Propgtion of B77 virus nd three strins of Rous srcom virus in Jpnese quil. Neoplsm 15: Theilen, G. H., R. Zeigel, nd M. J. Twiehus Biologicl studies with RE virus (strin T) tht induces reticuloendotheliosis in turkeys, chickens, nd Jpnese quil. J. Ntl. Cncer Inst. 37: Toyoshim, K., nd P. K. Vogt Enhncement nd inhibition of vin srcom viruses by polyctions nd polynions. Virology 38: Vogt, P. K A virus relesed by "nonproducing" Rous srcom cells. Proc. Ntl. Acd. Sci. U.S.A. 58: Vogt, P. K., nd R. R. Friis An vin leukosis virus relted to RSV(O): properties nd evidence for helper ctivity. Virology 43: Vogt, P. K., R. Ishizki, nd R. Duff Studies on the reltionships mong vin tumor viruses, p In Subvirl Crcinogenesis, Monogr. 1st Int. Symp. Tumor Viruses, Univ. of Colordo Medicl Center, Denver. 32. Weiss, R. A Interference nd neutrliztion studies with Bryn strin Rous srcom virus synthesized in the bsence of helper virus. J. Gen. Virol. 5: Downloded from on April 24, 2019 by guest