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Interactions Between Endogenous Virus Loci ev 6 and ev 21.
Interactions Between Endogenous Virus Loci ev6 and ev21. 2. Congenital Transmission of EV21 Viral Product to Female Progeny from Slow-Feathering Dams EUGENE J. SMITH, ALY M. FADLY, and LYMAN B. CRITTENDEN USDA, Agricultural Research Service, Regional Poultry Research Laboratory, 3606 East Mount Hope Road, East Lansing, Michigan 48823 (Received for publication November 21, 1989)
1990 Poultry Science 69:1251-1256 INTRODUCTION
Most chickens harbor multiple endogenous virus (ev) genes. Some ev genes produce infectious virus particles (EV), but some genes are defective in that only subviral genes such as envelope glycoproteins are expressed (Smith, 1987). Endogenous virus gene products can compromise the immune response to field strains of avian leukosis viruses (ALV) because the antigenic similarity between endogenous virus structural proteins and oncogenic field strains of ALV induces immunological tolerance toward ALV infection (Crittenden et al, 1982, 1984). The endogenous virus locus ev21 is genetically associated with the slow-feathering (SF) dominant gene K (Bacon et al, 1988). However, SF dams place their rapid-feathering (RF) (ev21~) progeny at risk to persistent ALV infection through congenital transmission of EV21 (Smith and Fadly, 1988). Congenital transmission of EV21 was shown to be eliminated in SF dams that are homozygous resistant at the tvb locus (Smith and Crittenden, 1988), but cellular resistance in the progeny requires that both parental lines must be homozygous tvb*. This report extends previous work (Smith et al, 1990) on the influence of ev21/ev6 on the
immune response to ALV infection and presents a selection strategy for reducing immunological tolerance to ALV infection induced by EV21. The ev21/ev6 interaction was of interest because defective ev genes such as ev6 interfere in ALV replication (Robinson et al, 1981), ev6 is widely distributed among White Leghorns, and ev6 is highly expressed (Hayward et al, 1980). In contrast to the previous report (Smith et al, 1990), chickens described here were rapidfeathering female progeny representative of a typical feather-sexed cross. Results focus on 1) the maternal influence of ev6 on congenital transmission of EV21, and 2) the immune response to ALV infection among ev6 + and ev6~ female progeny of SF and RF dams with and without ev6. MATERIALS AND METHODS
Chickens, Matings, and Exposure to RPL-40 Virus Pooled semen from specific pathogen-free RPL Line ISBl roosters homozygous susceptible to ALV A and E and homozygous for RF (ev21~) was used to inseminate SF (ev21+) and RF (ev21~) hens with and without ev6. Class 1
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ABSTRACT The influence of the endogenous virus ev6 on congenital transmission of EV21, the infectious viral product encoded by locus ev21, and the immune response to exogenous avian leukosis virus (ALV) infection was studied in rapid-feathering (RF) female progeny from four classes of slow-feathering (SF) (ev21 + and RF (ev21~) dams with and without ev6. Apart from transmitting infectious EV21 and ev6 to progeny, dam ev genotype did not influence the immune response or shedding of RPL-40. The endogenous virus envelope glycoprotein encoded by ev6, however, completely restricted shedding and congenital transmission of infectious endogenous virus EV21, from SF dams. After 19 wk of exposure to ALV strain RPL-40 infected cage mates, only 11% of the congenitaUy infected female progeny mounted neutralizing antibodies against RPL-40, whereas 73% of their noncongenitally infected sisters seroconverted. More ev6 female progeny, however, were shedders of RPL-40 and developed tumors than ev6~ sisters. Among progeny from the four classes of dams, EV21 congenitaUy infected hens had the highest incidence (31%) of RPL40-induced tumors. (Key words: viremia, antibodies, shedding, feathering genes, lymphomas)
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SMITH ET AL.
TABLE 1. Endogenous virus genotypes of dams and female progeny from four crosses with homozygous avian leucosis virus-susceptible rapid feathering Line 15B1 roosterr Progeny2 Dam Class
EV21
Shedders
Genotype
1-SF3
0/164
0/125
ev6*~lev6~ ev21+/H-
2-SF
15/15
12/12
ev6~/ev6~ ev21+fw ¥
0/10
0/8
ev6 /ev6~ ev21~/w
4-RF
0/8
0/7
ev6~/ev6~ ev21~/w
Congenitally infected
Genotype
1 1A
0/41 6 0/50
ev6*lev6~ ev6~/ev6~
2 2d
0/26 53/53
ev6~/ev6~ ev6~/ev6~
3 3A
0/4 0/5
ev&levfT ev6~/ev6~
4
0/20
ev6~lev6~
+
Sires were evl , ev6~, and ev21 . ^Responses to RFL-40 infection involved only rapid-feathering (ev21~) female progeny. The number of progeny from two hatches were pooled. 3 RF = rapid-feathering; SF = slow feathering. ''Number with EV21 (the infectious virus product encoded by ev21) in blood at hatch out of number tested. d u m b e r shedders of p27 in albumens out of number tested. d u m b e r of EV21 congenitally infected chicks at hatch out of number tested from each class of dams.
dams were ev6+/ev21+; Class 2 dams were ev6~/ ev21+; Class 3 dams were ev6+/ev21~; and Class 4 dams were ev6"7ev21~ (Table 1). As an indicator of shedding of EV21, prior to mating, one to three egg albumens from hens were tested for ALV-group specific antigen, p27, using an ELISA (Smith et al, 1979). All female progeny were susceptible to Subgroup E and carried evl but progeny from Class 1 and Class 3 dams were segregating for ev6. Two hatches were set 2 wk apart. The seven types of progeny from the four classes of dams were vaccinated with 2000 pfu of turkey herpesvirus and intermingled with day-old progeny from a RPL KI5 x 7j cross that were inoculated in the yolk sac as 7-day embryos with 10* infectious units of RPL-40 virus. The ratio of uninfected to RPL-40 virus-infected chickens was 5.3:1 and 6.6:1 for Hatches 1 and 2, respectively. Males from RF hens were removed from the experiment at 6 wk of age. Both flocks were euthanatized with carbon dioxide gas when chickens were 19 wk of age. Genotypes, Viremia, Antibody Response, and Shedding of RPL-40 About .2 mL of heparinized blood was drawn at hatch for EV21 assays and ev6 analysis (Smith and Crittenden, 1986). To determine whether
chicks were congenitally infected with EV21,. 1 ml of plasma from progeny of SF dams was placed in culture fluids of ALV Subgroup Esusceptible RPL Line 15B1 chick embryo fibroblasts (CEF). After 7 days of cultivation, the cell culture plates were frozen and thawed twice, and .1 mL of culture fluid was tested for ALV group-specific antigen p27 in an infectious ALV assay described by Crittenden et al. (1987). Ten and 19 wk after hatch, RPL-40 in plasmas was measured using Subgroup Eresistant RPL Line 0 (C/E) CEF, using the same protocol. The RPL-40 neutralizing antibodies (Ab) were examined in a microtiter assay using 50 jil of plasma (Fadly et al, 1989). Pathology All chicks that died, and those killed at 19 wk of age, were examined for gross lesions characteristic of ALV-induced tumors. Deaths not related to ALV infection were not included in the analysis. Statistical Analysis Significant differences in RPL-40 viremia, antibody response, cloacal shedding, and the incidence of tumors were determined by chisquare using 2 by 2 contingency tables.
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3-RF
Type
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ENDOGENOUS VIRUS LOCI ev6 AND ev21
TABLE 2. Influence of dam's ev67ev2i genotype on RPRL viremia (V), shedding (S), and antibody (Ab) response on evtf" female progeny 10 wk
Dam Class
1
2
Genotype
1. SF ev6+,ev21+ 2. SF ev6-,ev21+ 3. RF ev6 + ,ev21 4. RF ev6~,ev21~ Interactions (ev6 + ,cv21 + ) > (--) (-ev21 + ) > (cv6+-)
V 19/49 8/18 9/22 15/35
19 wk
Ab (39)3 (44) (41) (43)
34/84 (40) 17/40 (42)
31/49 9/17 18/25 20/35
V
S (63) (53) (72) (57)
51/84 (61) 27/42 (64)
25/45 8/15 16/25 21/39
(55) (53) (64) (54)
46/84 (54) 24/40 (60)
13/45 5/15 8/25 14/39
Ab (29) (33) (32) (36)
27/84 (32) 13/40 (32)
35/45 11/15 19/25 26/39
(77) (73) (76) (66)
61/84 (73) 30/40 (75)
*SF = slow-feathering; RF = rapid-feathering. No significant differences between dam's ev6/cv21 genotype and responses of progeny to RFL-40 infection were obtained using chi-square 2 by 2 contingency tables for all paired combinations. dumber positive out of number tested (%). 2
Role of ev<5 in Congenital Transmission of EV21 Egg albumens from the four classes of mature breeder hens were tested for the presence of ALV group-specific antigen, p27, prior to mating the hens with Line 15B1 roosters. All ev6+ SF hens tested that were not EV21 viremic at hatch were nonshedders of p27, whereas all ev6~ SF hens mat were EV21 viremic at hatch secreted detectable amounts of p27; p27 was not detected in albumens from RF dams (Table 1). From Class 2 dams that carried only ev21, 76 and 54% of their progeny from hatches 1 and 2, respectively, were congenitally (vertically) infected with EV21. The EV21 viral antigen was not detected in day-old progeny from dam Classes 3 and 4. Contact Spread of RPL-40 Infection To determine the rate of contact (horizontal) infection, blood was taken from flocks at 4 wk of age. Plasmas from 58 and 60 test chicks from Hatches 1 and 2, respectively, revealed that all chicks tested were infected with RFL-40 through contact with embryonally infected cage mates. Lack of Maternal Influence of ev6 on the Immune Response to RPL-40 Virus Infection Among ev6 Negative Progeny Apart from congenital transmission of EV21 and genetic transmission of ev6, no significant
differences in viremia, shedding, or antibody response were found among ev6~ progeny from each of the four classes of dams (Table 2). Moreover, no significant differences in the incidence of seroconversion or shedding were found when ev6~ progeny from dam Classes 1 and 4 were compared with progeny from dam Classes 2 and 3. Immune Response to RPL-40 Infection Among ev6 Positive Progeny When ev6+ and ev6~ progeny were compared, significantly more of the ev6+ progeny were found to be viremic with RPL-40 than their ev6~ hatchmates (Table 3). Although higher percentages of ev6+ progeny were persistently viremic and shedders of RPL-40, differences in the numbers of ev6 + and ev6~ progeny with regard to shedding or seroconversion were not significant when flocks were 19 wk of age. Role of Congenitally Transmitted EV21 on the Immune Response to RPL-40 Infection Analysis of RPL-40 viremia and antibody response at 10 and 19 wk of age for all seven types of progeny indicated a significant difference between hatches only when noncongenitally infected sisters of Type 2 were compared. Because only 18 progeny were in this category, hatches were pooled. Significantly more progeny that were congenitally infected with EV21 at hatch were persistently tolerant to RPL40 infection than noncongenitally infected progeny from Class 2 dams (Table 4). Of 37 congenitally infected progeny, only 11%
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RESULTS
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SMITH ET AL.
TABLE 3. Viremia (V), shedding (S), antibody (Ab) response, and RPL-40-induced tumors in rapid-feathering female progeny with and without ev6 10 wk Type
1
1 + 3 1A + 3A
ev6
V
19 wk S
Ab 3
+
a
41/71 (SS)" 41/71 (58) 28/71 (39)b 49/74 (66)a
V
Tumors2
Ab
a
a
a
44/68 (65) 26/69 (38) 47/68 (69) 41/70 (59)a 21/70 (30)a 54/70 (77)a
12/74 (16)a 4/84 (4) 6
^''Percentages within columns with no common superscripts are significantly different (P<05). 1 Type defines rapid feathering progeny from heterozygous eve"*" dams that segregated for ev6. 2 Cumulative number with rumors out of total number at risk. d u m b e r positive out of number tested (%), from two hatches.
TABLE 4. Influence of congenital transmission of EV21 (the infectious virus product encoded by ev21) on RPL-40 viremia (V), shedding (S), and antibody (Ab) response, and tumors1 10 wk Type 2
2a
V b
3
19 wk
Ab
8/18 (44) 34/39 (87)a
9/17 (53) 3/39 (8) 6
S a
8/15 (53)* 33/37 (89)a
V
Tumors2
Ab b
5/15 (33) 28/37 (76)a
a
11/15 (73) 4/37 ( l l ) b
^Percentages within columns with no common superscripts are significantly different (P<01). Number positive out of number tested (%), from two hatches. 2 Cumulative number with tumors out of total number at risk (%). 3 CI = congenitally infected with EV21.
2/16 (12) a 12/39 (31)a
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seroconverted, whereas 73% of noncongenitally Crossing RF roosters with SF dams is a infected sisters seroconverted within 19 wk after convenient feather-sexing strategy for producexposure to RPL-40 virus-infected hatchmates. tion of RF commercial egg layers. Even though RF progeny from this cross do not carry evil, congenital transmission of EV21 The Cumulative Incidence of Tumors from SF dams was also shown to compromise Among Progeny from Slow-Feathering the immune response to ALV infection (Smith and Rapid-Feathering Dams and Fadly, 1988). Persistent tolerance to Among progeny from Class 1 and Class 3 exogenous ALV infection in mature breeder dams, significantly more ev6+ progeny deve- hens would thus perpetuate the cycle of ALV loped tumors than ev6~ hatchmates (Table 3). infection among progeny of the next generaAlthough 5 or 46 (11%) females from Class 4 tion. dams also developed tumors, the difference in Two phenomena associated with the materthe inidence of tumors between ev6+ and ev6~ nal influence of ev6 and ev21 on congenital females was not significant (P>.10). Among transmission of EV21 and the immune reEV21 congenitally infected progeny, 12 of 39 sponse to ALV infection in experimental (31%) developed tumors characteristic of lymWhite Leghorn flocks were described here. It phoid leukosis (Table 4). was previously shown (Smith et al., 1990) that infectious EV21 and envelope glycoprotein DISCUSSION encoded at defective locus ev6 independently Early research showed that congenital infec- increased the incidence of persistent RPL-40 tion with ALV induces persistent viremia and viremia. However, EV21 was a more potent a debilitated humoral antibody response tolerogen than envelope glycoprotein. In the present study, the marked inhibition (Rubin et al., 1962). This immunologic tolerance is attributed to a failure to mount of congenital transmission of EV21 by enveneutralizing antibodies against viral envelope lope glycoprotein was evident when progeny glycoprotein (Qualtiere and Meyers, 1979). from Class 1 and Class 2 dams were com-
ENDOGENOUS VIRUS LOCI ev6 AND ev21
In ALV eradication programs, SF nonshedders of group-specific antigen (p27) are se-
lected as breeding stock. As shown here, ev6 plays a major role in inhibiting the replication, shedding, and congenital transmission of EV21 from SF dams to RF female progeny. The frequent occurrence of ev6 in commercial egg layers may reflect selection of nonshedders. In this context, Kuhnlein et al. (1989) recently reported an increase in the frequency of ev6 in four strains selected for egg production. In view of the close association between EV21 and K (Bacon et al, 1988), the possibility of selecting SF dams that lack ev21 appears remote. Selection for ev6+ breeder hens is problematic because envelope glycoproteins are tolerogenic. For poultry breeders, the benefits of eliminating congenital transmission of ev21 through ev6 interference may outweigh a moderate impairment in the immune response to ALV. To eliminate congenital transmission of EV21, selecting parents mat are homozygous resistant at the tvb locus represents one approach. The influence of ev6 on progeny from homozygousresistant parents, however, remains to be determined. ACKNOWLEDGMENTS
The authors thank John Gill and Dan Levin for advice on statistical analysis. The authors also appreciate the expert technical support of Randy Bonna, Cecyl Fischer, Leonard Provencher, and James Pulaski. REFERENCES Bacon, L. D., E. J. Smith, L. B. Crittenden, and G. B. Havenstein, 1988. Association of the slow feathering (K) and an endogenous vital {evil) gene on the Z chromosomes of chickens. Poultry Sci. 67:191-197. Crittenden, L. B., A. M. Fadly, and E. J. Smith, 1982. Effect of endogenous leukosis vims genes on response to infection with avian leukosis and reticuloendotheliosis viruses. Avian Dis. 26:279-294. Crittenden, L. B., S. McMahon, M. S. Halpern, and A. M. Fadly, 1987. Embryonic infection with the endogenous avian leukosis virus rous-associated virus-O alters responses to exogenous avian leukosis virus infection. J. Virol. 61:722-725. Crittenden, L. B., E. J. Smith, and A. M. Fadly, 1984. Influence of endogenous viral (ev) gene expression and strain of exogenous avian leukosis virus (ALV) on mortality and ALV infection and shedding in chickens. Avian Dis. 28:1037-1056. Fadly, A. M., T. F. Davison, L. N. Payne, and K. Howes, 1989. Avian leukosis virus infection and shedding in Brown Leghorn chickens treated with corticosterone or exposed to various stressors. Avian Pathol. 18: 283-298.
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pared. From combined hatches, all female progeny from Class 1 dams were nonviremic, whereas, 67% of the progeny from Class 2 dams were congenitally infected with EV21 (Table 1). The long-term (19 wk) consequence of congenital transmission was shown among RPL-40-exposed progeny of Class 2 dams; most of the congenitally infected hens (89%) were persistently tolerant to RPL-40 virus infection, as measured by shedding and neutralizing antibodies (Table 4). Conversely, 73% of their noncongenitally infected sisters seroconverted within 19 wk after exposure. The relatively high incidence of lymphomas in EV21 congenitally infected hens (31%), although not statistically significant, is consistent with an earlier report (Smith and Fadly, 1988). In experiments simulating congenital transmission of endogenous viruses, Crittenden et al. (1987) found that chickens embryonally infected with Rous-associated virus, Type O (RAV-O), and subsequently inoculated with RAV-1 and RAV-2, also had impaired antibody responses and higher incidence of lymphoid leukosis than hatchmates that were not inoculated as embryos. Although ev6+ dams did not congenitally transmit EV21, ev6 was genetically transmitted to about half of their progeny. Consequently, when flocks were 10 wk of age, significantly more ev6+ hens were viremic with RPL-40 than their ev6~ sisters. At 19 wk of age, although a higher percentage of ev6+ hens were still viremic, with RPL-40, significant differences in viremia between e\6+ and ev6~ hens were not found. Moreover, immunologic tolerance to RPL-40 virus induced by ev6 was not as marked or prolonged as that induced in hatchmates congenitally infected with EV21. hi earlier experiments, (Smith et al., 1990) more ev6+ hens were also persistently tolerant to RPL-40 than ev6~ sisters. The moderate tolerizing influence of ev6 relative to that of EV21, may reflect physicochemical differences in the structure of the gene products; EV21 is a fully infectious virus particle that is shed into the circulation, whereas envelope glycoproteins may remain largely bound to cell membranes or sequestered in a subcellular compartment. The higher incidence of tumors in ev6+ females compared with their ev6~ hatchmates is also consistent with the trend observed earlier.
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SMITH EI AL. Pages 101-120 in: Avian Leukosis. Martinus Nijhoff Publishing Co., Boston, MA. Smith, E. J., and L. B. Crittenden, 1986. Endogenous viral genes in a slow-feathering line of White Leghorn chickens. Avian Pathol. 15:395-406. Smith, E. J., and L. B. Crittenden, 1988. Genetic cellular resistance to subgroup E avian leukosis virus in slowfeathering dams reduces congenital transmission of an endogenous retrovirus encoded at locus ev21. Poultry Sci. 67:1668-1673. Smith, E. J., and A. M. Fadly, 1988. Influence of congenital transmission of endogenous virus-21 on the immune response to avian leukosis virus infection and the incidence of tumors in chickens. Poultry Sci. 67: 1674-1679. Smith, E. J., A. M. Fadly, and L. B. Crittenden, 1990. Interactions between endogenous virus loci ev6 and ev21.1. Immune response to exogenous avian leukosis virus infection. Poultry Sci. 69:1244-1250. Smith, E. J., A. Fadly, and W. Okazaki, 1979. An enzymelinked immunosorbent assay for detecting avian leukosis-sarcoma viruses. Avian Dis. 23:698-707.
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Hayward, W. S„ S. B. Braverman, and S. M. Astrin, 1980. Transcriptional products and DNA structure of endogenous avian proviruses. Cold Spring Harbor Symp. Quant. Biol. 44:1111-1122. Kuhnlein, U., M. Sabour, J. S. Gavora, R. W. Fairfull, and D. E.Bernon, 1989. Influence of selection for egg production and Marek's disease resistance on the incidence of endogenous viral genes in White Leghorns. Poultry Sci. 68:1161-1167. Qualtieie, L. F., and P. Meyers, 1979. A re-examination of humoral tolerance in chickens congenitally infected with an avian leukosis virus. J. Immunol. 122: 825-829. Robinson, H. L., S. M. Astrin, A. M. Senior, and F. H. Salazar, 1981. Host susceptibility to endogenous viruses: Defective, glycoprotein-expressing proviruses interfere with infections. J. Virol. 40:745-751. Rubin, H. L., L. Fanshier, A. Cornelius, and W. F. Hughes, 1962. Tolerance and immunity in chickens after congenital and contact infection with an avian leukosis virus. Virology 17:143-156. Smith, E. J., 1987. Endogenous avian leukemia viruses.
1990 Poultry Science 69:1251-1256 INTRODUCTION
Most chickens harbor multiple endogenous virus (ev) genes. Some ev genes produce infectious virus particles (EV), but some genes are defective in that only subviral genes such as envelope glycoproteins are expressed (Smith, 1987). Endogenous virus gene products can compromise the immune response to field strains of avian leukosis viruses (ALV) because the antigenic similarity between endogenous virus structural proteins and oncogenic field strains of ALV induces immunological tolerance toward ALV infection (Crittenden et al, 1982, 1984). The endogenous virus locus ev21 is genetically associated with the slow-feathering (SF) dominant gene K (Bacon et al, 1988). However, SF dams place their rapid-feathering (RF) (ev21~) progeny at risk to persistent ALV infection through congenital transmission of EV21 (Smith and Fadly, 1988). Congenital transmission of EV21 was shown to be eliminated in SF dams that are homozygous resistant at the tvb locus (Smith and Crittenden, 1988), but cellular resistance in the progeny requires that both parental lines must be homozygous tvb*. This report extends previous work (Smith et al, 1990) on the influence of ev21/ev6 on the
immune response to ALV infection and presents a selection strategy for reducing immunological tolerance to ALV infection induced by EV21. The ev21/ev6 interaction was of interest because defective ev genes such as ev6 interfere in ALV replication (Robinson et al, 1981), ev6 is widely distributed among White Leghorns, and ev6 is highly expressed (Hayward et al, 1980). In contrast to the previous report (Smith et al, 1990), chickens described here were rapidfeathering female progeny representative of a typical feather-sexed cross. Results focus on 1) the maternal influence of ev6 on congenital transmission of EV21, and 2) the immune response to ALV infection among ev6 + and ev6~ female progeny of SF and RF dams with and without ev6. MATERIALS AND METHODS
Chickens, Matings, and Exposure to RPL-40 Virus Pooled semen from specific pathogen-free RPL Line ISBl roosters homozygous susceptible to ALV A and E and homozygous for RF (ev21~) was used to inseminate SF (ev21+) and RF (ev21~) hens with and without ev6. Class 1
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ABSTRACT The influence of the endogenous virus ev6 on congenital transmission of EV21, the infectious viral product encoded by locus ev21, and the immune response to exogenous avian leukosis virus (ALV) infection was studied in rapid-feathering (RF) female progeny from four classes of slow-feathering (SF) (ev21 + and RF (ev21~) dams with and without ev6. Apart from transmitting infectious EV21 and ev6 to progeny, dam ev genotype did not influence the immune response or shedding of RPL-40. The endogenous virus envelope glycoprotein encoded by ev6, however, completely restricted shedding and congenital transmission of infectious endogenous virus EV21, from SF dams. After 19 wk of exposure to ALV strain RPL-40 infected cage mates, only 11% of the congenitaUy infected female progeny mounted neutralizing antibodies against RPL-40, whereas 73% of their noncongenitally infected sisters seroconverted. More ev6 female progeny, however, were shedders of RPL-40 and developed tumors than ev6~ sisters. Among progeny from the four classes of dams, EV21 congenitaUy infected hens had the highest incidence (31%) of RPL40-induced tumors. (Key words: viremia, antibodies, shedding, feathering genes, lymphomas)
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SMITH ET AL.
TABLE 1. Endogenous virus genotypes of dams and female progeny from four crosses with homozygous avian leucosis virus-susceptible rapid feathering Line 15B1 roosterr Progeny2 Dam Class
EV21
Shedders
Genotype
1-SF3
0/164
0/125
ev6*~lev6~ ev21+/H-
2-SF
15/15
12/12
ev6~/ev6~ ev21+fw ¥
0/10
0/8
ev6 /ev6~ ev21~/w
4-RF
0/8
0/7
ev6~/ev6~ ev21~/w
Congenitally infected
Genotype
1 1A
0/41 6 0/50
ev6*lev6~ ev6~/ev6~
2 2d
0/26 53/53
ev6~/ev6~ ev6~/ev6~
3 3A
0/4 0/5
ev&levfT ev6~/ev6~
4
0/20
ev6~lev6~
+
Sires were evl , ev6~, and ev21 . ^Responses to RFL-40 infection involved only rapid-feathering (ev21~) female progeny. The number of progeny from two hatches were pooled. 3 RF = rapid-feathering; SF = slow feathering. ''Number with EV21 (the infectious virus product encoded by ev21) in blood at hatch out of number tested. d u m b e r shedders of p27 in albumens out of number tested. d u m b e r of EV21 congenitally infected chicks at hatch out of number tested from each class of dams.
dams were ev6+/ev21+; Class 2 dams were ev6~/ ev21+; Class 3 dams were ev6+/ev21~; and Class 4 dams were ev6"7ev21~ (Table 1). As an indicator of shedding of EV21, prior to mating, one to three egg albumens from hens were tested for ALV-group specific antigen, p27, using an ELISA (Smith et al, 1979). All female progeny were susceptible to Subgroup E and carried evl but progeny from Class 1 and Class 3 dams were segregating for ev6. Two hatches were set 2 wk apart. The seven types of progeny from the four classes of dams were vaccinated with 2000 pfu of turkey herpesvirus and intermingled with day-old progeny from a RPL KI5 x 7j cross that were inoculated in the yolk sac as 7-day embryos with 10* infectious units of RPL-40 virus. The ratio of uninfected to RPL-40 virus-infected chickens was 5.3:1 and 6.6:1 for Hatches 1 and 2, respectively. Males from RF hens were removed from the experiment at 6 wk of age. Both flocks were euthanatized with carbon dioxide gas when chickens were 19 wk of age. Genotypes, Viremia, Antibody Response, and Shedding of RPL-40 About .2 mL of heparinized blood was drawn at hatch for EV21 assays and ev6 analysis (Smith and Crittenden, 1986). To determine whether
chicks were congenitally infected with EV21,. 1 ml of plasma from progeny of SF dams was placed in culture fluids of ALV Subgroup Esusceptible RPL Line 15B1 chick embryo fibroblasts (CEF). After 7 days of cultivation, the cell culture plates were frozen and thawed twice, and .1 mL of culture fluid was tested for ALV group-specific antigen p27 in an infectious ALV assay described by Crittenden et al. (1987). Ten and 19 wk after hatch, RPL-40 in plasmas was measured using Subgroup Eresistant RPL Line 0 (C/E) CEF, using the same protocol. The RPL-40 neutralizing antibodies (Ab) were examined in a microtiter assay using 50 jil of plasma (Fadly et al, 1989). Pathology All chicks that died, and those killed at 19 wk of age, were examined for gross lesions characteristic of ALV-induced tumors. Deaths not related to ALV infection were not included in the analysis. Statistical Analysis Significant differences in RPL-40 viremia, antibody response, cloacal shedding, and the incidence of tumors were determined by chisquare using 2 by 2 contingency tables.
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3-RF
Type
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ENDOGENOUS VIRUS LOCI ev6 AND ev21
TABLE 2. Influence of dam's ev67ev2i genotype on RPRL viremia (V), shedding (S), and antibody (Ab) response on evtf" female progeny 10 wk
Dam Class
1
2
Genotype
1. SF ev6+,ev21+ 2. SF ev6-,ev21+ 3. RF ev6 + ,ev21 4. RF ev6~,ev21~ Interactions (ev6 + ,cv21 + ) > (--) (-ev21 + ) > (cv6+-)
V 19/49 8/18 9/22 15/35
19 wk
Ab (39)3 (44) (41) (43)
34/84 (40) 17/40 (42)
31/49 9/17 18/25 20/35
V
S (63) (53) (72) (57)
51/84 (61) 27/42 (64)
25/45 8/15 16/25 21/39
(55) (53) (64) (54)
46/84 (54) 24/40 (60)
13/45 5/15 8/25 14/39
Ab (29) (33) (32) (36)
27/84 (32) 13/40 (32)
35/45 11/15 19/25 26/39
(77) (73) (76) (66)
61/84 (73) 30/40 (75)
*SF = slow-feathering; RF = rapid-feathering. No significant differences between dam's ev6/cv21 genotype and responses of progeny to RFL-40 infection were obtained using chi-square 2 by 2 contingency tables for all paired combinations. dumber positive out of number tested (%). 2
Role of ev<5 in Congenital Transmission of EV21 Egg albumens from the four classes of mature breeder hens were tested for the presence of ALV group-specific antigen, p27, prior to mating the hens with Line 15B1 roosters. All ev6+ SF hens tested that were not EV21 viremic at hatch were nonshedders of p27, whereas all ev6~ SF hens mat were EV21 viremic at hatch secreted detectable amounts of p27; p27 was not detected in albumens from RF dams (Table 1). From Class 2 dams that carried only ev21, 76 and 54% of their progeny from hatches 1 and 2, respectively, were congenitally (vertically) infected with EV21. The EV21 viral antigen was not detected in day-old progeny from dam Classes 3 and 4. Contact Spread of RPL-40 Infection To determine the rate of contact (horizontal) infection, blood was taken from flocks at 4 wk of age. Plasmas from 58 and 60 test chicks from Hatches 1 and 2, respectively, revealed that all chicks tested were infected with RFL-40 through contact with embryonally infected cage mates. Lack of Maternal Influence of ev6 on the Immune Response to RPL-40 Virus Infection Among ev6 Negative Progeny Apart from congenital transmission of EV21 and genetic transmission of ev6, no significant
differences in viremia, shedding, or antibody response were found among ev6~ progeny from each of the four classes of dams (Table 2). Moreover, no significant differences in the incidence of seroconversion or shedding were found when ev6~ progeny from dam Classes 1 and 4 were compared with progeny from dam Classes 2 and 3. Immune Response to RPL-40 Infection Among ev6 Positive Progeny When ev6+ and ev6~ progeny were compared, significantly more of the ev6+ progeny were found to be viremic with RPL-40 than their ev6~ hatchmates (Table 3). Although higher percentages of ev6+ progeny were persistently viremic and shedders of RPL-40, differences in the numbers of ev6 + and ev6~ progeny with regard to shedding or seroconversion were not significant when flocks were 19 wk of age. Role of Congenitally Transmitted EV21 on the Immune Response to RPL-40 Infection Analysis of RPL-40 viremia and antibody response at 10 and 19 wk of age for all seven types of progeny indicated a significant difference between hatches only when noncongenitally infected sisters of Type 2 were compared. Because only 18 progeny were in this category, hatches were pooled. Significantly more progeny that were congenitally infected with EV21 at hatch were persistently tolerant to RPL40 infection than noncongenitally infected progeny from Class 2 dams (Table 4). Of 37 congenitally infected progeny, only 11%
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RESULTS
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SMITH ET AL.
TABLE 3. Viremia (V), shedding (S), antibody (Ab) response, and RPL-40-induced tumors in rapid-feathering female progeny with and without ev6 10 wk Type
1
1 + 3 1A + 3A
ev6
V
19 wk S
Ab 3
+
a
41/71 (SS)" 41/71 (58) 28/71 (39)b 49/74 (66)a
V
Tumors2
Ab
a
a
a
44/68 (65) 26/69 (38) 47/68 (69) 41/70 (59)a 21/70 (30)a 54/70 (77)a
12/74 (16)a 4/84 (4) 6
^''Percentages within columns with no common superscripts are significantly different (P<05). 1 Type defines rapid feathering progeny from heterozygous eve"*" dams that segregated for ev6. 2 Cumulative number with rumors out of total number at risk. d u m b e r positive out of number tested (%), from two hatches.
TABLE 4. Influence of congenital transmission of EV21 (the infectious virus product encoded by ev21) on RPL-40 viremia (V), shedding (S), and antibody (Ab) response, and tumors1 10 wk Type 2
2a
V b
3
19 wk
Ab
8/18 (44) 34/39 (87)a
9/17 (53) 3/39 (8) 6
S a
8/15 (53)* 33/37 (89)a
V
Tumors2
Ab b
5/15 (33) 28/37 (76)a
a
11/15 (73) 4/37 ( l l ) b
^Percentages within columns with no common superscripts are significantly different (P<01). Number positive out of number tested (%), from two hatches. 2 Cumulative number with tumors out of total number at risk (%). 3 CI = congenitally infected with EV21.
2/16 (12) a 12/39 (31)a
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seroconverted, whereas 73% of noncongenitally Crossing RF roosters with SF dams is a infected sisters seroconverted within 19 wk after convenient feather-sexing strategy for producexposure to RPL-40 virus-infected hatchmates. tion of RF commercial egg layers. Even though RF progeny from this cross do not carry evil, congenital transmission of EV21 The Cumulative Incidence of Tumors from SF dams was also shown to compromise Among Progeny from Slow-Feathering the immune response to ALV infection (Smith and Rapid-Feathering Dams and Fadly, 1988). Persistent tolerance to Among progeny from Class 1 and Class 3 exogenous ALV infection in mature breeder dams, significantly more ev6+ progeny deve- hens would thus perpetuate the cycle of ALV loped tumors than ev6~ hatchmates (Table 3). infection among progeny of the next generaAlthough 5 or 46 (11%) females from Class 4 tion. dams also developed tumors, the difference in Two phenomena associated with the materthe inidence of tumors between ev6+ and ev6~ nal influence of ev6 and ev21 on congenital females was not significant (P>.10). Among transmission of EV21 and the immune reEV21 congenitally infected progeny, 12 of 39 sponse to ALV infection in experimental (31%) developed tumors characteristic of lymWhite Leghorn flocks were described here. It phoid leukosis (Table 4). was previously shown (Smith et al., 1990) that infectious EV21 and envelope glycoprotein DISCUSSION encoded at defective locus ev6 independently Early research showed that congenital infec- increased the incidence of persistent RPL-40 tion with ALV induces persistent viremia and viremia. However, EV21 was a more potent a debilitated humoral antibody response tolerogen than envelope glycoprotein. In the present study, the marked inhibition (Rubin et al., 1962). This immunologic tolerance is attributed to a failure to mount of congenital transmission of EV21 by enveneutralizing antibodies against viral envelope lope glycoprotein was evident when progeny glycoprotein (Qualtiere and Meyers, 1979). from Class 1 and Class 2 dams were com-
ENDOGENOUS VIRUS LOCI ev6 AND ev21
In ALV eradication programs, SF nonshedders of group-specific antigen (p27) are se-
lected as breeding stock. As shown here, ev6 plays a major role in inhibiting the replication, shedding, and congenital transmission of EV21 from SF dams to RF female progeny. The frequent occurrence of ev6 in commercial egg layers may reflect selection of nonshedders. In this context, Kuhnlein et al. (1989) recently reported an increase in the frequency of ev6 in four strains selected for egg production. In view of the close association between EV21 and K (Bacon et al, 1988), the possibility of selecting SF dams that lack ev21 appears remote. Selection for ev6+ breeder hens is problematic because envelope glycoproteins are tolerogenic. For poultry breeders, the benefits of eliminating congenital transmission of ev21 through ev6 interference may outweigh a moderate impairment in the immune response to ALV. To eliminate congenital transmission of EV21, selecting parents mat are homozygous resistant at the tvb locus represents one approach. The influence of ev6 on progeny from homozygousresistant parents, however, remains to be determined. ACKNOWLEDGMENTS
The authors thank John Gill and Dan Levin for advice on statistical analysis. The authors also appreciate the expert technical support of Randy Bonna, Cecyl Fischer, Leonard Provencher, and James Pulaski. REFERENCES Bacon, L. D., E. J. Smith, L. B. Crittenden, and G. B. Havenstein, 1988. Association of the slow feathering (K) and an endogenous vital {evil) gene on the Z chromosomes of chickens. Poultry Sci. 67:191-197. Crittenden, L. B., A. M. Fadly, and E. J. Smith, 1982. Effect of endogenous leukosis vims genes on response to infection with avian leukosis and reticuloendotheliosis viruses. Avian Dis. 26:279-294. Crittenden, L. B., S. McMahon, M. S. Halpern, and A. M. Fadly, 1987. Embryonic infection with the endogenous avian leukosis virus rous-associated virus-O alters responses to exogenous avian leukosis virus infection. J. Virol. 61:722-725. Crittenden, L. B., E. J. Smith, and A. M. Fadly, 1984. Influence of endogenous viral (ev) gene expression and strain of exogenous avian leukosis virus (ALV) on mortality and ALV infection and shedding in chickens. Avian Dis. 28:1037-1056. Fadly, A. M., T. F. Davison, L. N. Payne, and K. Howes, 1989. Avian leukosis virus infection and shedding in Brown Leghorn chickens treated with corticosterone or exposed to various stressors. Avian Pathol. 18: 283-298.
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pared. From combined hatches, all female progeny from Class 1 dams were nonviremic, whereas, 67% of the progeny from Class 2 dams were congenitally infected with EV21 (Table 1). The long-term (19 wk) consequence of congenital transmission was shown among RPL-40-exposed progeny of Class 2 dams; most of the congenitally infected hens (89%) were persistently tolerant to RPL-40 virus infection, as measured by shedding and neutralizing antibodies (Table 4). Conversely, 73% of their noncongenitally infected sisters seroconverted within 19 wk after exposure. The relatively high incidence of lymphomas in EV21 congenitally infected hens (31%), although not statistically significant, is consistent with an earlier report (Smith and Fadly, 1988). In experiments simulating congenital transmission of endogenous viruses, Crittenden et al. (1987) found that chickens embryonally infected with Rous-associated virus, Type O (RAV-O), and subsequently inoculated with RAV-1 and RAV-2, also had impaired antibody responses and higher incidence of lymphoid leukosis than hatchmates that were not inoculated as embryos. Although ev6+ dams did not congenitally transmit EV21, ev6 was genetically transmitted to about half of their progeny. Consequently, when flocks were 10 wk of age, significantly more ev6+ hens were viremic with RPL-40 than their ev6~ sisters. At 19 wk of age, although a higher percentage of ev6+ hens were still viremic, with RPL-40, significant differences in viremia between e\6+ and ev6~ hens were not found. Moreover, immunologic tolerance to RPL-40 virus induced by ev6 was not as marked or prolonged as that induced in hatchmates congenitally infected with EV21. hi earlier experiments, (Smith et al., 1990) more ev6+ hens were also persistently tolerant to RPL-40 than ev6~ sisters. The moderate tolerizing influence of ev6 relative to that of EV21, may reflect physicochemical differences in the structure of the gene products; EV21 is a fully infectious virus particle that is shed into the circulation, whereas envelope glycoproteins may remain largely bound to cell membranes or sequestered in a subcellular compartment. The higher incidence of tumors in ev6+ females compared with their ev6~ hatchmates is also consistent with the trend observed earlier.
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SMITH EI AL. Pages 101-120 in: Avian Leukosis. Martinus Nijhoff Publishing Co., Boston, MA. Smith, E. J., and L. B. Crittenden, 1986. Endogenous viral genes in a slow-feathering line of White Leghorn chickens. Avian Pathol. 15:395-406. Smith, E. J., and L. B. Crittenden, 1988. Genetic cellular resistance to subgroup E avian leukosis virus in slowfeathering dams reduces congenital transmission of an endogenous retrovirus encoded at locus ev21. Poultry Sci. 67:1668-1673. Smith, E. J., and A. M. Fadly, 1988. Influence of congenital transmission of endogenous virus-21 on the immune response to avian leukosis virus infection and the incidence of tumors in chickens. Poultry Sci. 67: 1674-1679. Smith, E. J., A. M. Fadly, and L. B. Crittenden, 1990. Interactions between endogenous virus loci ev6 and ev21.1. Immune response to exogenous avian leukosis virus infection. Poultry Sci. 69:1244-1250. Smith, E. J., A. Fadly, and W. Okazaki, 1979. An enzymelinked immunosorbent assay for detecting avian leukosis-sarcoma viruses. Avian Dis. 23:698-707.
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Hayward, W. S„ S. B. Braverman, and S. M. Astrin, 1980. Transcriptional products and DNA structure of endogenous avian proviruses. Cold Spring Harbor Symp. Quant. Biol. 44:1111-1122. Kuhnlein, U., M. Sabour, J. S. Gavora, R. W. Fairfull, and D. E.Bernon, 1989. Influence of selection for egg production and Marek's disease resistance on the incidence of endogenous viral genes in White Leghorns. Poultry Sci. 68:1161-1167. Qualtieie, L. F., and P. Meyers, 1979. A re-examination of humoral tolerance in chickens congenitally infected with an avian leukosis virus. J. Immunol. 122: 825-829. Robinson, H. L., S. M. Astrin, A. M. Senior, and F. H. Salazar, 1981. Host susceptibility to endogenous viruses: Defective, glycoprotein-expressing proviruses interfere with infections. J. Virol. 40:745-751. Rubin, H. L., L. Fanshier, A. Cornelius, and W. F. Hughes, 1962. Tolerance and immunity in chickens after congenital and contact infection with an avian leukosis virus. Virology 17:143-156. Smith, E. J., 1987. Endogenous avian leukemia viruses.