JOURNAL OF VIROLOGY, Oct. 1967, p. 898-904Copyright © 1967 American Society for Microbiology

Vol. 1, No. 5Printed in U.S.A.

Two Loci Controlling Genetic Cellular Resistance to

Avian Leukosis-Sarcoma VirusesLYMAN B. CRITTENDEN, HOWARD A. STONE, RICHARD H. REAMER, AND WILLIAM OKAZAKI

Regional Poultry Research Laboratory, U. S. Department of Agriculture, East Lansing, Michigan 48823

Received for publication 20 April 1967

Female chickens known to be heterozygous for resistance to subgroups A and Bof the avian leukosis-sarcoma viruses were mated to males known to be homozy-gously resistant to both. The progeny were assayed both on the chorioallantoicmembrane (CAM) and in tissue culture for resistance to representative viruses ofthe A, B, and tentatively defined C subgroups. Segregation ratios of resistance toA and B subgroup viruses agreed with the previously suggested hypothesis ofsingle-autosomal-recessive genes controlling resistance to each subgroup. Mixedinfection on the CAM and replicate plate infection in tissue culture with subgroupA and B viruses showed that resistance to the A and B subgroups was inherited inde-pendently. Assays with viruses tentatively classified as subgroup C indicated thatthey were largely composed of a mixture of subgroup A and B viruses or of particlespossessing the host range specificity of both. However, virus stocks of the subgroupC category, as well as some stocks classified as subgroup B, produced small numbersof pocks or foci on individuals known to be resistant to subgroup A and B viruses.It is suggested that these Rous sarcoma virus stocks carry between 1 and 10% of atrue subgroup C virus.

The avian leukosis-sarcoma viruses may beclassified into at least three subgroups (A, B, andC), based on properties of the virus coat. Theseproperties are host range, interference patterns,and antigenic type (6, 14, 15; Vogt et al., Sym-posium on Subviral Carcinogenesis, Nagoya,Japan, in press).A single-autosomal-recessive gene controls

specific cellular resistance to the in vitro and invivo growth of subgroup A viruses (1-3, 8). Simi-larly, a second single-autosomal-recessive genecontrols resistance to subgroup B viruses (10,11). It is clear that resistance to these viral sub-groups is independent at the phenotypic level,because all four combinations of resistance orsusceptibility are found (14). However, no studieshave utilized critical matings which would revealwhether these genes are linked or independent.Preliminary data from this laboratory suggestthat these genes are, in fact, independently in-herited (R. H. Reamer, unpublished data). Thepresent study was designed specifically to test thishypothesis, by inoculation of back-cross progenyfrom matings designed to segregate in a one-to-one ratio of resistance to susceptibility to bothsubgroups A and B with Rous sarcoma viruses(RSV) representative of these subgroups.The same progeny were also inoculated with

viruses tentatively classified as subgroup C in anattempt to identify resistance to this subgroup.

MATERIALS AND METHODS

Phenotypic and genotypic nomenclature. Vogt and'Ishizaki (14) suggested a phenotypic nomenclaturebased on the terminology for host bacterium resistanceused by bacteriophage workers. We suggested (3)genotypic terminology, using Rs and rs for gene sym-bols representing an abbreviation for RSV. Since it isnow known that these genes control resistance toleukosis viruses as well as to RSV, and since it issuspected that at least two loci are involved, we haveadopted the terminology presented in Table 1. Wecall these loci the "tumor virus a" (tva) and the"tumor virus b" (tvb) loci, specifying susceptibilitypatterns to the avian tumor virus subgroups A and B,respectively. The superscripts r and s stand for re-sistance and susceptibility, respectively, and multiplealleles may be designated by numbered superscripts.

Virus stocks. Table 2 gives the virus stocks used,their subgroup classification, and their abbreviations.The Bryan high-titer pseudotypes BH-RSV(RAV-1)

and BH-RSV(RAV-2) were obtained from P. K. Vogt.These stocks were originally produced by activation ofnonvirus-producing transformed (NP) cells withRAV-1 or RAV-2 which had been "cloned" by limitdilution (14). The BH-RSV (RAV-1) seed virus waspropagated once by passage in the pectoral muscle ofline 151 isolated chickens (7); BH-RSV(RAV-2) was

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LOCI FOR AVIAN LEUKOSIS-SARCOMA VIRUSES

TABLE 1. Genotypic and phenotypic nomenclaturefor genetic cellular resistance to the avian

leukosis-sarcoma viruses controlled bythe tva and tvb loci

Susceptibility (S) orresistance (R) to avian

Genotype Phenotype tumor virus subgroup

A B

as as bs bs C/O S S

as ar ba bs C/O S Sar ar bs bs C/A R Sas a8 bs br C/O S Sas ar bs br C/O S Sar ar bs br C/A R Sa8 as br br C/B S Ra, ar br br C/B S Ra r ar b r br CA,B R R

propagated in line 7, known to be resistant to sub-group A viruses. The tumor minces were suspended in0.15 M potassium citrate with 1 mg of hyalurc;nidaseper 100 ml, homogenized in a Waring B'endor, andclarified by centrifugation at 2,000 X g in an Inter-national refrigerated centrifuge.The Schmidt-Ruppin stocks, SR-RSV-1 and SR-

RSV-2, were prepared from heterogeneous materialobtained from Padman Sarma (12). These stockswere selected for A and B subgroup specificity bygrowing them for five consecutive passages of super-natant fluid in C/B and C/A cells, respectively. Asecond preparation of SR-RSV-2 was obtained fromP. K. Vogt. This was passaged on C/A cells andclassified as predominantly subgroup B. These stockswere propagated in line 151 or line 7 chickens for one

passage as indicated above.Since it was apparent, at least in tissue culture, that

the resistance to B viruses could be overcome rathereasily, the relative titers of several of the viruses were

determined in C/O and C/A,B embryos and in tissueculture. Tenfold dilutions of the virus preparationswere inoculated into the cells or embryos of line 6 and7. The logarithm (base 10) of the titers in line 6 (C/O),line 7 (C/A,B), and the difference between them are

given for each virus in Table 3. Substantial differencesof at least three logs were observed in both assaysystems on inoculation with both subgroup A viruses.

The subgroup B viruses showed a different picture.Large differences in titer were observed on inoculationwith BH-RSV(RAV-2). However, the differences intiter in both systems were substantially lower whentwo different sources of SR-RSV-2 were used. There-fore, the unexpected susceptibility to these viruses ofembryos carrying the b' allele in the homozygousstate must be attributed to the virus stocks used andnot to the assay method. For this reason, difficultywas experienced in identifying the C/B and C/A,Bphenotypes unequivocally when SR-RSV-2 was used.The BS-RSV was prepared as described by Purchase

and Okazaki (10). The HA-RSV preparation has beendescribed by Crittenden and Okazaki (1). Both ofthese stocks have demonstrated their ability to dif-ferentiate among phenotypes on chorioallantoicmembrane (CAM) assay (1).

Matings. Inbred lines 6 and 7, and their F, andback-cross matings, were used in this study. Extensivedata have shown that line 7 is homozygously resistant(ar ar), line 6 is homozygously susceptible (a' a'), andthe back-cross progeny of an F, X line 7 mating segre-

gates in a ratio of one resistant to one susceptible inresponse to inoculation with BS-RSV, which is pre-

dominantly subgroup A (1, 3, 14). Table 4 presents thesusceptibility of these lines upon inoculation withHA-RSV and PR-RSV. It is clear from these data andother unpublished data (Crittenden) that line 6 ishomozygously susceptible to HA-RSV (b8 be), whereasline 7 is segregating for resistance to this subgroup Bvirus. However, subline 2 of line 7 appears to behomozygously resistant (br br) to this subgroup. Thissame segregation pattern is seen with PR-RSV, sug-

gesting a possible relationship with subgroup B.Thirty-nine F, females from reciprocal cross matingsof lines 6 and 7 were available for study. Since thesubline origin of the line 7 parent was unknown, thesedams could have been double heterozygotes (a' ar,bs bt) or homozygously susceptible to subgroup Bwhile heterozygous for susceptibility to subgroup A(ta' ar, b bR). These females were mated to males ofsubline 2 of line 7, known to be ar a,, br bt. A prelimi-nary test of these matings by inoculation on the CAMwith BH-RSV(RAV-2) revealed 24 which producedat least two resistant progeny. These 24 matings were

assumed to be the double back-cross or test-crossmatings appropriate for one test of independentsegregation. That is, if these genes are independent,the two parental (C/O, C/A,B) and the two recombi-

TABLE 2. Stocks of virus used and their subgroup identity according to Vogt anzd Ishizaki (14)

SubgroupStock

A B C or unknown

Bryan high-titer RSV pseudotypes BH-RSV (RAV-1) BH-RSV (RAV-2)Schmidt-Ruppin RSV SR-RSV-1 SR-RSV-2Bryan Standard RSV BS-RSVHarris RSV HA-RSVCarr-Zilber RSV CZ-RSVPrague RSV PR-RSV

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CRITTENDEN ET AL. J. VIROL.TABLE 3. Differences in titer-s (log1o) of pairs of subgroup A and B virus preparationts in line 6

(CIO) and lin7e 7 (C/A, B) embryos whenl assayed in tissue culture anid on the CAM

Assay

Tissue culture assayLine 6 (C/O) titer.

Line 7 (C/A, B) titer....

Difference.......................

CAM assayLine 6 (C/O) titer...............

Line 7 (C/A, B) titer...

Difference.

Subgroup A

BH-RSV(RAV-1)

6.62

<1.00

>5.62

6.58

3.20

3.38

SR-RSV-1

6.00

< 2.00

> 4.00

5.93

1.70

4.23

Subgroup B

BH-RSV(RAV/-2) SR-RSV-2

6.1la 6.236.00 4.88

6.30< 2.00 4.042.00 3.26

3.54>4.11 2.194.00 1.62

2.76

5.62 5.804.Ilb

2.36 4.912.95

3.26 0.891.16

a More than one entry means that the titration was repeated.I The second entry represents virus obtained from P. K. Vogt and propagated by us.

TABLE 4. Tabulationi of acpproximate pock counits onthe embryonic clorioallalntoic membranes of linles6 an2d 7 and their cross after inioculationt of

HA -RSV anid PR-RSV

Line

66 X 7

7

67

Sub-line

AllAll1234

All1234

Inoculum

HA-RSVHA-RSVHA-RSVHA-RSVHA-RSVHA-RSV

PR-RSVPR-RSVPR-RSVPR-RSVPR-RSV

a Estimates over 20.

Pock counta

O 1-20 21-50 1l 100+ Total100

0 0 2 11 18 310 0 1 26 33 60

23 4 9 25 23 8479 5 0 0 0 843 1 35 82 95 216

25 0 1 7 1 34

31124

0

280

0

40

0

100

211

0

5

0

1004

0

11

0

1261019280

nant (C/A, C/B) phenotypes should occur in equalfrequency.

Assay procedures. The CAM and tissue cultureassay procedures were essentially those previouslydescribed (3) and follow the procedures given byGroupe et al. (4) and Temin and Rubin (13).

RESULTS

CAM assays. Twenty-two test-cross matingswere used for the CAM assay experiments. The

10

10 DILUTION

50 10t 150 200Number of Pocks

FIG. 1. Distribution of pock counts ont the CAM oftest-cross embryos inzoculated with two dilutions ofBH-RSV(RAV-I). CAM numbers were grouiped fortabulation in successive five-pock categories. Datarepresent one of three settings of eggs. Pocks becameconfluent when more than 300 were present; therefore,all membranes showing 300 or more pocks were tabu-lated as 300+.

eggs from these matings were incubated in 10consecutive biweekly settings. In each setting, agroup of line 6 (C/O) and line 7 (C/A,B) eggswere set as known susceptible and resistant con-trols, and these behaved as expected. Four dif-ferent inocula were used in concentrations whichwould give an average of at least 100 pocks per

900

0.a rA- ~~~~~~V91;

I711111111111111111

II

ILI]

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TABLE 5. Segregationt ratios of test-cross embryossusceptible and resistant to four inocula of

Rous sarcoma virus

Sex No. No.Virus Test-cross mating of sus- resis-ae X (ol X 9) em- cep- tant

bryo tibie

BH-RSV (RAV-1) 7 X (7 X 6) e 33 367 X (7 X 6) 9 46 387X (6X7) e 43 467X (6X7) 9 51 46Total 173 166

BH-RSV (RAV-2) 7 X (7 X 6) e 45 657 X (7 X 6) 9 65 537 X (6 X 7) e 52 637X (6X7) 9 51 59Total 213 240

BH-RSV (RAV-1) 7 X (7 X 6) e 37 11mixed with BH- 7 X (7 X 6) 9 34 7RSV (RAV-2) 7X (6X7) e 37 14

7 X (6 X 7) 9 42 19Total 150 51

PR-RSV 7X (7X6) dP 50 97X (7X6) 9 33 117 X (6 X 7) d' 50 167X (6X7) 9 46 13Total 179 49

membrane. The sex of each embryo was deter-mined when the pocks were counted.

Representative distributions of pock counts forembryos inoculated with BH-RSV(RAV-1) aregiven in Fig. 1. The 10-3 dilution was used toprovide most of the segregation data collected inthree settings. Any embryo inoculated with thisdilution showing four or fewer pocks was con-sidered resistant. It is clear that the distributionsare bimodal and that it was not difficult to differ-entiate between resistant and susceptible em-bryos. The segregation data are given in Table 5.These data are grouped by sex of the embryoand the reciprocal mating status of the dam inorder to evaluate any possible influence of sexlinkage. The fact that chi-square tests showed nosignificant heterogeneity of segregation ratiosamong the four mating type-sex groups indicatedthat this locus is not on the sex chromosome.The ratio of 173 susceptible to 166 resistant em-bryos agrees very well with the 1 :1 ratio expectedfor subgroup A viruses, assuming that suscepti-bility is controlled by a single-autosomal-domi-nant gene.

Similar results with BH-RSV(RAV-2) wereobtained (Fig. 2 and Table 5). Although thesegregation ratios are not as close to 1:1 as inthe case of BH-RSV(RAV-1), they still agree

20[

I0

20

z

10

1.2 x 10-' DILUTION

Blrl1.2 x 10' DILUTION

VARci0 50 100 150 200 250

Number of Pocks

300+

FIG. 2. Distribution ofpock counlts on the CAM oftest-cross embryos inoculated with two dilutions ofBH-RSV(RA V-2).

with the single-autosomal-gene hypothesis forthe control of resistance to subgroup B viruses.The next inoculum used was a mixture of

BH-RSV(RAV-1) and BH-RSV (RAV-2) in suchconcentrations that each virus alone should haveproduced an average count of at least 100 pocks.If the tva and tvb genes were at the same locus orclosely linked, one would expect only the parentalC/O and C/A,B phenotypes in equal frequency.However, if tva and tvb were located at unlinkedloci, the recombinant C/A and C/B phenotypeswould be expected in frequency equal to theparental C/O and C/A, B phenotypes. This wouldgive a 3:1 ratio of susceptible and partiallysusceptible (C/O, C/A, and C/B) to resistant(C/A,B) phenotypes. This method of testing forlinkage in the RSV system was first used by Payneand Biggs (9). Again, the segregation ratios werefound to be homogeneous (Table 5). The veryclose fit to a 3:1 ratio of susceptible to resistantembryos shows that these two single-gene sys-tems are not at closely linked loci. Figure 3gives the distribution of pock counts for one of

a 20

E 10

-. L j-a

0 50 100 150 200 250 300

Number of Packs

FIG. 3. Distribution of pock counzts on the CAMof test-cross embryos inoculated with a mixture ofBH-RSV(RA V-I) and BH-RSV(RA V-2). Data repre-seiit one of two settings of eggs.

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TABLE 6. Mediatn and rantge offocus cOulnts onl conitrol liine 6 anzd linle 7 cells inioculated withsix strains of RSV ini 14 trials

- Subgroup A Subgroup B Subgroup CLine Phenotype mination

I SR-RS'-1l BS-RSV-1 SR-RSV-2 HA-RSV PR-RSV CZ-RSV

6 C/O Median 2,180 3,000+a 2,380 3,000+ 3,000+ 3,000+Range 520-3,000+ 600-3,000+ 740-(3,000+ 1,110-3,000+ 1,100-3,000+ 1,520-3,000+

7 C/A, B Median 0 0 0 0 0 0Range 0-0 0-0 0-450 0-120 0-40 0-360

a Pocks became confluent when counts were more than 3,000 per 60-ml plastic dish.

the two settings inoculated with the mixture. Itis clear that the distribution is bimodal, but noclear distinction can be made between the C/Oand selectively resistant (C/A and C/B) pheno-types.Vogt and Ishizaki (15) suggested that PR-RSV

belongs to a third or C subgroup. Since theC/A,B subline of line 7 and line 6 (C/O) werefound to be resistant and susceptible to theseviruses, these same matings were appropriate forstudying segregation ratios with this virus stock.Again, segregation ratios were found to behomogeneous, and a 3:1 ratio of susceptible toresistant embryos was observed (Table 5). Thissuggests that susceptibility to this virus stock iscontrolled by two independent loci, perhaps ivaand tvb.

Tissue culture assay. Secondary cell cultures ofa single embryo may be inoculated with differentvirus stocks in separate plates to estimate theembryo phenotype. This is an obvious advantagewhen more than one virus subgroup is involved.The test-cross embryos from four F1 dams were

used for tissue culture assay. Primary cell cul-tures were made each week from 10 test-crossembryos, one line 6 (C/O) embryo, and one line 7(C/A,B) embryo. Replicate secondary cultureswere inoculated in 60-mm plastic plates witheach of six virus preparations in concentrationswhich would give an average of at least 1,000 fociper plate. Two plates from each embryo wereheld as uninoculated controls. The median andrange of focus counts on line 6 and 7 controlplates for the 14 trials are given in Table 6. Ineach case, the subgroup A viruses produced highfocus counts on the line 6 (C/O) and no foci onthe line 7 (C/A, B) cell sheets. However, fociwere noticed on some of the C/A,B cell sheetswhen inoculated with subgroup B and C viruses.The significance of these foci will be consideredin the Discussion. It was arbitrarily decided tocall embryos with 10%- or less of the line 6 con-trol count resistant for the purpose of classifyinga culture.Table 7 gives the phenotypic classification of

TABLE 7. Phenotypes of test-cross embryos, basedonI susceptibility to SR-RSV-I and SR-RSV-2

o;ily, with 10% of the linte 6 focus coutnts asthe cr'iterionl of' resistanice

Dam C/o C/A C/B C/A,B Total

18 15 5 3 9 3222 8 10 7 8 3325 4 10 5 10 2936 13 8 7 5 33

Total 40 33 22 32 127

127 test-cross embryos based on the suscepti-bility of embryo tissue cultures to SR-RSV-1 andSR-RSV-2 (stocks produced at this laboratory).Inoculation with SR-RSV-1 gave a ratio of 62susceptible (C/O and C/B) to 65 resistant (C/Aand C/A,B), an excellent fit to the expected 1:1segregation at the tva locus. For SR-RSV-2, theratio was 73 susceptible (C/O and C/A) to 54resistant (C/B and C/A,B). This ratio is notsignificantly different from 1:1. The ratio of 72parental (C/O, C/AB) to 55 recombinant (C/A,C/B) phenotypes, while showing an excess ofparental phenotypes, is not significantly differentfrom the 1:1 ratio expected for independentsegregation.To investigate the possibility that genes inde-

pendent of the tva and tvb loci control a cell sur-face property specific for the proposed subgroupC viruses, a composite tabulation of the cellularphenotypes from all six inocula was made. Suffi-cient information was available for classificationof 122 embryos. Again, the criterion used to de-fine resistance was a plating efficiency of 10% orless compared with the line 6 controls. The in-volvement of three independent loci in the geneticcellular resistance seen in our material would leadto the appearance of eight cellular phenotypes inequal frequencies. However, only four phenotypesoccurred in appreciable frequency (Table 8).The phenotypes for susceptibility and resistanceto the subgroup A and B viruses segregatedapproximately 1 :1 and were independent as

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TABLE 8. Cell phenotypes of test-cross embryos determined by use of 10% of the line 6 focuscounts as the criterion of resistanice for all six virus stocks

Dam C/O C/A C/B C/C C/A, B C/A, C C/B, C C/A, B, C Total

18 15 4 3 1 0 0 0 8 3122 8 10 7 0 0 1 2 4 3225 4 10 5 0 1 0 0 9 2936 12 5 5 0 1 1 1 5 30

Total 39 29 20 1 2 2 3 26 122

before. However, the response to the proposedsubgroup C viruses gave a segregation ratio of 90susceptible to 31 resistant, a 3:1 ratio, as wasfound on the CAM assay with PR-RSV. Further-more, subgroup C susceptibility is clearly de-pendent on both the tva and tvb loci. That is, thecells susceptible to either A or B subgroups (orboth) are usually susceptible to subgroup C. The3 :1 ratio observed is a consequence of this de-pendence.As pointed out in the previous section, it was

sometimes difficult to identify the cellular pheno-types with certainty. We believe that the cases inwhich presumptive subgroup C susceptibility orresistance appears to be independent of the pheno-types controlled by the tva and tvb loci representerrors of classification.

DIsCUSSION

Both the CAM and tissue culture assay systemsconfirm that two single-autosomal-dominantgenes control susceptibility to subgroup A and Bviruses, respectively. There is no evidence of sexlinkage because the segregation of progeny of F1dams produced by reciprocal matings of lines 6and 7 was 1:1. Also, male and female progenyeach gave 1 :1 ratios of susceptibility to resistanceon CAM assay. Results of both methods of assayindicate that the genes controlling susceptibilityto subgroup A and B viruses are inherited inde-pendently, showing that the tva and tvb loci arenot closely linked.CZ-RSV and PR-RSV have been tentatively

placed in subgroup C (15). The present data indi-cate that the ability of these viruses to infect cellsis influenced by both the tva and tvb loci. Thissuggests that these virus preparations are approxi-mately equal mixtures of subgroup A and B vi-ruses or that single particles possess protein coatproperties complementary to cell surface sitesfor attachment or entry of both subgroup A andB viruses. The latter could occur either by geneticrecombination of the subgroup A and B con-trolling elements so that they occur in one parti-cle, or by production of protein envelopes withboth specificities by dual infection with subgroupA and B viruses, that is, phenotypic mixing. In-

vestigations of cloned virus stocks are necessaryto differentiate among these several alternatives.Recent studies by Vogt (Virology, in press) showthat phenotypic mixing among two subgroups,A and B, does occur in the avian tumor viruses.A small proportion of embryos appeared to

have phenotypes for susceptibility to subgroup Cviruses independent of the tva and tvb loci. It isunlikely that these are due to close linkage of atvc locus to the other two loci, for two reasons.First, a segregation of three susceptible embryosto one resistant embryo is observed after inocula-tion with these viruses, whereas a 1 :1 ratio wouldbe expected if a single tvc locus controlled re-sistance. Second, if a third locus were closelylinked to the tva and tvb loci, these also shouldshow evidence of linkage with each other. Theydo not. It is most likely that the exceptionalphenotypes occurred through misclassification,but the possibility of their existence should notbe ignored. It is clear, then, that at least the majorportions of the CZ-RSV and PR-RSV stocks areprobably not of a third subgroup, C, at least byhost range criteria.The successful infection of C/A,B cells and

embryos, particularly with subgroup B viruses,raises the question of the degree of resistancecontrolled by these loci. At least two alternativesto true infection of C/A ,B cells with subgroup Aor B viruses exist. One is that some viable cellsare present in the inoculum and produce foci orpocks through transplantation and virus produc-tion. This is possible because the inocula werecentrifuged but not filtered or subjected to ultra-sonic vibration. However, each inoculum did gothrough at least two cycles of freezing and thaw-ing. It seems unlikely that the presence of viablecells is the full explanation, becausethemethods ofpreparation of the two SR-RSV-2 stocks werevery similar to the methods used for the stockshowing larger differences in titer between theC/O and C/A,B phenotypes (Table 3). The factthat difference in titer found with the relativelypure stock of BH-RSV(RAV-2) was much greaterthan in the case of SR-RSV-2 suggests that par-ticularly the SR-RSV, PR-RSV, and CZ-RSVstocks may contain 1 to 10% of a true subgroup C

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virus which does grow in C/A, B cells. Observa-tions by Vogt, Ishizaki, and Duff (Proceedings ofthe Symposium on Subviral Carcinogenesis,Nagoya, Japan, in press) confirm the presence ofa large proportion of subgroup A and B virusesin the PR-RSV and CZ-RSV stocks. Further-more, they found evidence for a small proportionof a virus plating on C/A ,B cells in these stocks.The fact that RAV-50 was isolated from SR-RSV(5) and has been shown to be largely subgroup Csuggests that our stocks of SR-RSV-2 could havebeen mixed with virus of this subgroup. There-fore, it seems most likely that viral mixturesexplain the unexpected susceptibility of C/Bembryos and cells.

LITERATURE CITED1. CRIFTENDEN, L. B., AND W. OKAZAKI. 1965.

Genetic influence of the Rs locus on susceptibil-ity to avian tumor viruses. I. Neoplasms in-duced by RPL12 and three strains of Roussarcoma virus. J. Natl. Cancer Inst. 35:857-863.

2. CRITTENDEN, L. B., AND W. OKAZAKI. 1966.Genetic influence of the Rs locus on susceptibil-ity to avian tumor viruses. II. Rous sarcomavirus antibody production after strain RPL12virus inoculation. J. Natl. Cancer Inst. 36:299-303.

3. CRITTIENDEN, L. B., W. OKAZAKI, AND R. H.REAMER. 1964. Genetic control of responses toRous sarcoma and strain RPL12 viruses inthe cells, embryos, and chickens of two inbredlines. Nat]. Cancer Inst. Monograph 17:161-177.

4. GROUPE, V., V. C. DUNKEL, AND R. A. MANAKER.1957. Improved pock counting method for thetitration of Rous sarcoma virus in embryonatedeggs. J. Bacteriol. 74:409-410.

5. HANAFUSA, H., AND T. HANAFUSA. 1966. Deter-mining factor in the capacity of Rous sarcomavirus to produce tumors in mammals. Proc.Natl. Acad. Sci. U. S. 55:532-538.

6. ISHIZAKI, R., AND P. K. VOGT. 1966. Immunologi-cal relationships among envelope antigens ofavian tumor viruses. Virology 30:375-387.

7. OKAZAKI, W. 1964. Discussion. Natl. CancerInst. Monograph 17:504-507.

8. PAYNE, L. N., AND P. M. BIGGS. 1964. Differencesbetween highly inbred lines of chickens in theresponse to Rous sarcoma virus of the chorioal-lantoic membrane and of embryonic cells intissue culture. Virology 24:610-616.

9. PAYNE, L. N., AND P. M BIGGS. 1966. Geneticbasis of cellular susceptibility to the Schmidt-Ruppin and Harris strains of Rous sarcomavirus. Virology 29:190-198.

10. PURCHASE, H. G., AND W. OKAZAKI. 1964. Mor-phology of foci produced by standard prepara-tions of Rous sarcoma virus. J. Natl. CancerInst. 32:579-589.

11. RUBIN, H. 1965. Genetic control of cellularsusceptibility to pseudotypes of Rous sarcomavirus. Virology 26:270-276.

12. SARMA, P. S., R. J. HUEBNER, AND D. ARMSTRONG.1964. A simplified tissue culture tube neutraliza-tion test for Rous sarcoma virus antibodies.Proc. Soc. Exptl. Biol. Med. 115:481-486.

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